Signal boost cascade assay

ABSTRACT

The present disclosure relates to compositions of matter and assay methods used to detect one or more target nucleic acids of interest in a sample. The compositions and methods provide signal boost upon detection of target nucleic acids of interest in less than one minute and in some instances instantaneously at ambient temperatures down to 16° C. or less, without amplification of the target nucleic acids yet allowing for massive multiplexing, high accuracy and minimal non-specific signal generation.

RELATED APPLICATIONS

This application claims priority to U.S. Ser. No. 63/289,112, filed 13 Dec. 2021; U.S. Ser. No. 63/359,183, filed 07 Jul. 2022; U.S. Ser. No. 63/395,394, filed 05 Aug. 2022; and U.S. Ser. No. 63/397,785, filed 12 Aug. 2022.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

Submitted herewith is an electronically filed sequence listing via EFS-Web a Sequence Listing XML, entitled “LS004US1_seqlist_20221201”, created 01 Dec. 2022, which is 1,227,000 bytes in size. The sequence listing is part of the specification of this specification and is incorporated by reference in its entirety.

PETITION UNDER 37 CFR 1.84(A)(2)

This patent application contains at least one drawing executed in color. The color drawings are necessary as the only practical medium by which aspects of the claimed subject matter may be accurately conveyed. The claimed invention relates to variant proteins that alter the active site thereof and the color drawings are necessary to easily discern the structural difference between variants. As the color drawings are being filed electronically via EFS-Web, only one set of the drawings is required.

FIELD OF THE INVENTION

The present disclosure relates to compositions of matter and assay methods used to detect one or more target nucleic acids of interest in a sample. The compositions and methods provide a signal boost upon detection of target nucleic acids of interest in less than one minute and at ambient temperatures down to 16° C. or less.

BACKGROUND OF THE INVENTION

In the following discussion certain articles and methods will be described for background and introductory purposes. Nothing contained herein is to be construed as an “admission” of prior art. Applicant expressly reserves the right to demonstrate, where appropriate, that the articles and methods referenced herein do not constitute prior art under the applicable statutory provisions.

Rapid and accurate identification of, e.g., infectious agents, microbe contamination, variant nucleic acid sequences that indicate the present of diseases such as cancer or contamination by heterologous sources is important in order to select correct treatment; identify tainted food, pharmaceuticals, cosmetics and other commercial goods; and to monitor the environment including identification of biothreats. Classic PCR and nucleic acid-guided nuclease or CRISPR (clustered regularly interspaced short palindromic repeats) detection methods rely on pre-amplification of target nucleic acids of interest to enhance detection sensitivity. However, amplification increases time to detection and may cause changes to the relative proportion of nucleic acids in samples that, in turn, lead to artifacts or inaccurate results. Improved technologies that allow very rapid and accurate detection of nucleic acids are therefore needed for timely diagnosis and treatment of disease, to identify toxins in consumables and the environment, as well as in other applications.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will be apparent from the following written Detailed Description including those aspects illustrated in the accompanying drawings and defined in the appended claims.

The present disclosure provides compositions of matter and assay methods to detect target nucleic acids of interest. The “nucleic acid-guided nuclease cascade assays” or “signal boost cascade assays” or “cascade assays” described herein comprise two different ribonucleoprotein complexes and either blocked nucleic acid molecules or blocked primer molecules. The blocked nucleic acid molecules or blocked primer molecules keep one of the ribonucleoprotein complexes “locked” unless and until a target nucleic acid of interest activates the other ribonucleoprotein complex. The present nucleic acid-guided nuclease cascade assay can detect one or more target nucleic acids of interest (e.g., DNA, RNA and/or cDNA) at attamolar (aM) (or lower) limits in less than one minute and in some embodiments virtually instantaneously without the need for amplifying the target nucleic acid(s) of interest, thereby avoiding the drawbacks of multiplex DNA amplification, such as primer-dimerization. Further, the cascade assay prevents “leakiness” that can lead to non-specific signal generation resulting in false positives by preventing unwinding of the blocked nucleic acid molecules or blocked primer molecules (double-stranded molecules); thus, the cascade assay is quantitative in addition to being rapid. A particularly advantageous feature of the cascade assay is that, with the exception of the gRNA in RNP1, the cascade assay components are the same in each assay no matter what target nucleic acid(s) of interest is being detected; moreover, the gRNA in the RNP1 is easily reprogrammed using traditional guide design methods.

The present disclosure is related first, to the instantaneous cascade assay, and second, to three modalities for preventing any “leakiness” in the cascade assay leading to false positives. The three modalities enhance the cascade assay and are in addition to using blocked nucleic acid molecules or blocked primer molecules in the cascade assay.

A first embodiment provides a method for identifying a target nucleic acid of interest in a sample in one minute or less at 16° C. or more comprising the steps of: providing a reaction mixture comprising: first ribonucleoprotein complexes (RNP1s) each comprising a first nucleic acid-guided nuclease and a first gRNA, wherein the first gRNA comprises a sequence complementary to the target nucleic acid of interest; and wherein binding of the RNP1 complex to the target nucleic acid of interest activates cis-cleavage and trans-cleavage activity of the first nucleic acid-guided nuclease; second ribonucleoprotein complexes (RNP2s) comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest; wherein the second nucleic acid-guided nuclease optionally comprises a variant nuclease engineered such that single stranded DNA is cleaved faster than double stranded DNA is cleaved, wherein the variant nuclease comprises at least one mutation to the domains that interact with the PAM region or surrounding sequences on blocked nucleic acid molecules, and wherein the variant nuclease exhibits both cis- and trans-cleavage activity; a plurality of the blocked nucleic acid molecules comprising a sequence corresponding to the second gRNA, wherein the blocked nucleic acid molecules comprise: a first region recognized by the RNP2 complex; one or more second regions not complementary to the first region forming at least one loop; one or more third regions complementary to and hybridized to the first region forming at least one clamp, wherein the plurality of blocked nucleic acid molecules and the RNP2s optionally are at a concentration ratio where the blocked nucleic acid molecules are at an equal or higher molar concentration than the RNP2s in the reaction mixture, wherein the blocked nucleic acid molecules optionally each comprise at least one bulky modification, and wherein the reaction mixture comprises at least one of a variant nuclease, the concentration ratio of the blocked nucleic acid molecules at a higher molar concentration than the molar concentration of RNP2s in the reaction mixture, and/or the blocked nucleic acid molecules comprise at least one bulky modification; contacting the reaction mixture with the sample under conditions that allow the target nucleic acid of interest in the sample to bind to RNP1, wherein upon binding of the target nucleic acid of interest RNP1 becomes active initiating trans-cleavage of at least one of the plurality of blocked nucleic acid molecules thereby producing at least one unblocked nucleic acid molecule, and wherein the at least one unblocked nucleic acid molecule binds to RNP2 initiating trans-cleavage of at least one further blocked nucleic acid molecule; and detecting the cleavage products, thereby detecting the target nucleic acid of interest in the sample in one minute or less.

An additional embodiment provides a method for identifying a target nucleic acid of interest in a sample in one minute or less at 16° C. or more comprising the steps of: providing a reaction mixture comprising: first ribonucleoprotein complexes (RNP1s), wherein the RNPls comprise a first nucleic acid-guided nuclease and a first guide RNA (gRNA); wherein the first gRNA comprises a sequence complementary to the nucleic acid target of interest, and wherein the first nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; second ribonucleoprotein complexes (RNP2s) comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest; wherein the second nucleic acid-guided nuclease optionally comprises a variant nuclease engineered such that single stranded DNA is cleaved faster than double stranded DNA is cleaved, wherein the variant nuclease comprises at least one mutation to the domains that interact with the PAM region or surrounding sequences on a synthesized activating molecule, and wherein the variant nuclease exhibits both cis- and trans-cleavage activity; a plurality of template molecules comprising sequence homology to the second gRNA; a plurality of the blocked primer molecules comprising a sequence complementary to the template molecules, wherein the blocked primer molecules cannot be extended by a polymerase, and wherein the blocked primer molecules comprise: a first region recognized by the RNP2; one or more second regions not complementary to the first region forming at least one loop; and one or more third regions complementary to and hybridized to the first region forming at least one clamp, wherein the plurality of blocked primer molecules and the RNP2s optionally are at a concentration ratio where the blocked nucleic acid molecules are at a higher molar concentration than the RNP2s in the reaction mixture, wherein the blocked primer molecules each optionally comprise at least one bulky modification, and wherein the reaction mixture comprises at least one of a variant nuclease, a concentration ratio where the blocked nucleic acid molecules are at a higher molar concentration than the RNP2s in the reaction mixture, and/or the blocked nucleic acid molecules comprising at least one bulky modification; and a polymerase and a plurality of nucleotides; contacting the reaction mixture with the sample under conditions that allow nucleic acid targets of interest in the sample to bind to RNP1, wherein: upon binding of the nucleic acid targets of interest to the RNP1, the RNP1 becomes active trans-cleaving at least one of the blocked primer molecules, thereby producing at least one unblocked primer molecule that can be extended by the polymerase; the at least one unblocked primer molecule binds to one of the template molecules and is extended by the polymerase and nucleotides to form at least one synthesized activating molecule having a sequence complementary to the second gRNA; and the at least one synthesized activating molecule binds to the second gRNA, and RNP2 becomes active cleaving at least one further blocked primer molecule and at least one reporter moiety in a cascade; allowing the cascade to continue; and detecting the unblocked primer molecules, thereby detecting the target nucleic acid of interest in the sample in one minute or less.

Aspects of the embodiments of the methods for identifying a target nucleic acid of interest in a sample in one minute or less can be substituted for any assay for identifying target nucleic acids; for example, for detecting human pathogens; animal pathogens; disease biomarkers; pathogens in laboratories, food processing facilities, hospitals, and in the environment, including bioterrorism applications (see the exemplary organisms listed in Tables 1, 2, 3, 5 and 6 and the exemplary human biomarkers listed in Table 4). Suitable samples for testing include any environmental sample, such as air, water, soil, surface, food, clinical sites and products, industrial sites and products, pharmaceuticals, medical devices, nutraceuticals, cosmetics, personal care products, agricultural equipment and sites, and commercial samples, and any biological sample obtained from an organism or a part thereof, such as a plant, animal (including humans), or microbe.

There is also provided in an embodiment a method of detecting a target nucleic acid molecule in a sample in a cascade reaction comprising the steps of: (a) providing a reaction mixture comprising: (i) a first ribonucleoprotein complex (RNP1) comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA) comprising a sequence complementary to a target nucleic acid molecule; (ii) a second ribonucleoprotein complex (RNP2) comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid molecule; and (iii) a plurality of blocked nucleic acid molecules comprising a sequence complementary to the second guide RNA, (b) contacting the target nucleic acid molecule with the reaction mixture under conditions that, relative to a control reaction, reduce the probability of R-loop formation between the second gRNA and the plurality of blocked nucleic acid molecules, wherein: (i) upon binding of the target nucleic acid molecule, the RNP1 becomes active wherein the first nucleic acid-guided nuclease cleaves at least one of the blocked nucleic acid molecules, thereby producing at least one unblocked nucleic acid molecule; and (ii) at least one unblocked nucleic acid molecule binds to the second gRNA, and the RNP2 becomes active wherein the second nucleic acid-guided nuclease cleaves at least one further blocked nucleic acid molecule; and (c) detecting the cleavage products of step (b), thereby detecting the target nucleic acid molecule in the sample.

There is also provided a second embodiment comprising a method of increasing the efficiency, reducing the background, increasing the signal-to-noise ratio, reducing cis-cleavage of blocked nucleic acid molecules and preventing unwinding of the second ribonucleoprotein complex (RNP2) in a cascade reaction comprising: (a) a reaction mixture comprising: (i) a first ribonucleoprotein complex (RNP1) comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA) comprising a sequence complementary to a target nucleic acid molecule; (ii) the RNP2 comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid molecule; and (iii) a plurality of blocked nucleic acid molecules comprising a sequence complementary to the second guide RNA, and (b) the target nucleic acid molecule comprising a sequence complementary to the first gRNA; and the method comprising the step of initiating the cascade reaction by contacting (a) and (b) under conditions that reduce the probability of R-loop formation between the blocked nucleic acid molecules and the second gRNA, thereby reducing increasing the efficiency, reducing the background, increasing the signal-to-noise ratio, reducing cis-cleavage of blocked nucleic acid molecules and preventing unwinding of the RNP2 relative to a control reaction.

There is also provided in a third embodiment a method of increasing the signal-to-noise ratio in a cascade reaction comprising the steps of: (a) providing a reaction mixture comprising: (i) a first ribonucleoprotein complex (RNP1) comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA) comprising a sequence complementary to a target nucleic acid molecule; (ii) a second ribonucleoprotein complex (RNP2) comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid molecule; and (iii) a plurality of blocked nucleic acid molecules comprising a sequence complementary to the second guide RNA, (b) initiating the cascade reaction by contacting the target nucleic acid molecule with the reaction mixture under conditions that reduce the probability of R-loop formation between the second gRNA and the plurality of blocked nucleic acid molecules, thereby increasing the signal-to-noise ratio in the cascade reaction relative to a control reaction, wherein: (i) upon binding of the target nucleic acid molecule, the RNP1 becomes active cleaving at least one of the blocked nucleic acid molecules, thereby producing at least one unblocked nucleic acid molecule; and (ii) the least one unblocked nucleic acid molecule binds to the second gRNA, and the RNP2 becomes active cleaving at least one further blocked nucleic acid molecule; and (c) detecting the cleavage products of the cascade reaction in step (b); and (d) determining the signal-to-noise ratio of the cascade reactions in step (b).

A fourth embodiment provides a method of increasing the efficiency, reducing the background, increasing the signal-to-noise ratio, reducing cis-cleavage of blocked nucleic acid molecules and preventing unwinding of a second ribonucleoprotein complex (RNP2) in a cascade reaction comprising the steps of: (a) providing a reaction mixture comprising: a first ribonucleoprotein complex (RNP1) comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA) comprising a sequence complementary to a target nucleic acid molecule; the RNP2 comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid molecule; and a plurality of blocked nucleic acid molecules comprising a sequence complementary to the second guide RNA, (b) initiating the cascade reaction by contacting the target nucleic acid molecule with the reaction mixture under conditions that reduce the probability of R-loop formation between the second gRNA and the plurality of blocked nucleic acid molecules, thereby increasing the efficiency, reducing the background, increasing the signal-to-noise ratio, reducing cis-cleavage of blocked nucleic acid molecules and preventing unwinding of the RNP2 in the cascade reaction relative to a control reaction.

In some aspects of these embodiments, the conditions that reduce R-loop formation comprise one or more of the steps of: 1) providing a molar concentration of blocked nucleic acid molecules that exceeds the molar concentration of ribonucleoprotein complexes; 2) engineering the nucleic acid-guided nuclease used in the ribonucleoprotein complex to result in a variant nucleic acid-guided nuclease such that single stranded DNA is cleaved faster than double stranded DNA is cleaved; and/or 3) engineering the blocked nucleic acid molecules to include bulky modifications of a size of about 1 nm or less.

Another embodiment provides a method for preventing unwinding of blocked nucleic acid molecules in the presence of an RNP in a cascade reaction comprising the steps of: providing blocked nucleic acid molecules; providing ribonucleoprotein complexes comprising a nucleic acid-guided nuclease that exhibits both cis- and trans-cleavage activity upon activation and a gRNA that recognizes an unblocked nucleic acid molecule resulting from trans-cleavage of the blocked nucleic acid molecules; and providing a molar concentration of the blocked nucleic acid molecules that exceeds the molar concentration of ribonucleoprotein complexes; engineering the nucleic acid-guided nuclease used in the ribonucleoprotein complex to result in a variant nucleic acid-guided nuclease such that single stranded DNA is cleaved faster than double stranded DNA is cleaved; and/or 3) engineering the blocked nucleic acid molecules to include bulky modifications of a size of about 1 nm or less thereby preventing unwinding of the blocked nucleic acid molecules in the cascade reaction.

In some aspects of the aforementioned embodiments, the blocked nucleic acid molecules are blocked primer molecules.

In a further embodiment, there is provided a method for preventing unwinding of blocked nucleic acid molecules or blocked primer molecules in the presence of an RNP comprising the steps of: providing blocked nucleic acid molecules or blocked primer molecules; providing ribonucleoprotein complexes comprising a nucleic acid-guided nuclease that exhibits both cis- and trans-cleavage activity upon activation and a gRNA that recognizes an unblocked nucleic acid molecule or an unblocked primer molecule resulting from trans-cleavage of the blocked nucleic acid molecule or blocked primer molecule; and providing a molar concentration of blocked nucleic acid molecules that exceeds the molar concentration of ribonucleoprotein complexes; engineering the nucleic acid-guided nuclease used in the ribonucleoprotein complex to result in a variant nucleic acid-guided nuclease such that single stranded DNA is cleaved times faster than double stranded DNA is cleaved; and/or 3) engineering the blocked nucleic acid molecules to include bulky modifications of a size of about 1 nm or less.

Other embodiments provide a method for detecting target nucleic acid molecules in a sample in less than one minute without amplifying the target nucleic acid molecules; and instantaneously detecting target nucleic acid molecules in a sample without amplifying the target nucleic acid molecules.

In some aspects of the methods, the reaction mixture is provided at 16° C., and in some aspects, the reaction mixture is provided at 17° C., 18° C., 19° C., 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., or 30° C. or higher.

Other embodiments provide reaction mixtures for identifying a target nucleic acid of interest in a sample in one minute or less comprising: first ribonucleoprotein (RNP1) complexes (RNP1s) each comprising a first nucleic acid-guided nuclease and a first gRNA, wherein the first gRNA comprises a sequence complementary to the target nucleic acid of interest; and wherein binding of the RNP1 complex to the target nucleic acid of interest activates cis-cleavage and trans-cleavage activity of the first nucleic acid-guided nuclease; second ribonucleoprotein complexes (RNP2s) comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest; wherein the second nucleic acid-guided nuclease optionally comprises a variant nuclease engineered such that single stranded DNA is cleaved faster than double stranded DNA is cleaved, wherein the variant nuclease comprises at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules, and wherein the variant nuclease exhibits both cis- and trans-cleavage activity; and a plurality of the blocked nucleic acid molecules comprising a sequence corresponding to the second gRNA, wherein the blocked nucleic acid molecules comprise: a first region recognized by the RNP2 complex; one or more second regions not complementary to the first region forming at least one loop; one or more third regions complementary to and hybridized to the first region forming at least one clamp, and wherein the blocked nucleic acid molecules optionally each comprise at least one bulky modification, wherein the plurality of blocked nucleic acid molecules and the RNP2s optionally are at a concentration ratio where blocked nucleic acid molecules are at a higher molar concentration than the RNP2s in the reaction mixture, and wherein the reaction mixture comprises at least one of a variant nuclease, a concentration ratio where blocked nucleic acid molecules are at a higher molar concentration than the RNP2s in the reaction mixture, and/or blocked nucleic acid molecules comprising at least one bulky modification.

Also provided is a reaction mixture for identifying a target nucleic acid of interest in a sample in one minute or less comprising: first ribonucleoprotein complexes (RNP1s), wherein the RNPls comprise a first nucleic acid-guided nuclease and a first guide RNA (gRNA); wherein the first gRNA comprises a sequence complementary to the nucleic acid target of interest, and wherein the first nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; second ribonucleoprotein complexes (RNP2s) comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest; wherein the second nucleic acid-guided nuclease optionally comprises a variant nuclease engineered such that single stranded DNA is cleaved faster than double stranded DNA is cleaved, wherein the variant nuclease comprises at least one mutation to the domains that interact with the PAM region or surrounding sequences on synthesized activating molecules, and wherein the variant nuclease exhibits both cis- and trans-cleavage activity; a plurality of template molecules comprising sequence homology to the second gRNA; a plurality of the blocked primer molecules comprising a sequence complementary to the template molecules, wherein the blocked primer molecules cannot be extended by a polymerase, and wherein the blocked primer molecules comprise: a first region recognized by the RNP2; one or more second regions not complementary to the first region forming at least one loop; and one or more third regions complementary to and hybridized to the first region forming at least one clamp, wherein the blocked primer molecules optionally each comprise at least one bulky modification and wherein the plurality of blocked primer molecules and the RNP2s optionally are at a concentration ratio where blocked nucleic acid molecules are at a higher molar concentration than the RNP2s in the reaction mixture, and wherein the reaction mixture comprises at least one of a variant nuclease, at a concentration ratio where blocked nucleic acid molecules are at a higher molar concentration than the RNP2s in the reaction mixture, and/or blocked nucleic acid molecules comprising at least one bulky modification; and a polymerase and a plurality of nucleotides.

Further provided is a composition of matter comprising: ribonucleoprotein complexes (RNPs) comprising a nucleic acid-guided nuclease and a gRNA that is not complementary to the target nucleic acid of interest; wherein the nucleic acid-guided nuclease optionally comprises a variant nuclease engineered such that single stranded DNA is cleaved faster than double stranded DNA is cleaved, wherein the variant nuclease comprises at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules, and wherein the variant nuclease exhibits both cis- and trans-cleavage activity; and a plurality of the blocked nucleic acid molecules comprising a sequence corresponding to the gRNA, wherein the blocked nucleic acid molecules comprise: a first region recognized by the RNP complex; one or more second regions not complementary to the first region forming at least one loop; one or more third regions complementary to and hybridized to the first region forming at least one clamp, wherein the blocked nucleic acid molecules each comprise at least one bulky modification, wherein the blocked nucleic acid molecules optionally each comprise at least one bulky modification, and wherein the plurality of blocked nucleic acid molecules and the RNP2s optionally are at a concentration ratio where the blocked nucleic acid molecules are at a higher molar concentration than the RNP2s in the reaction mixture, and wherein the composition comprises at least one of a variant nuclease, a concentration ratio where the blocked nucleic acid molecules are at a higher molar concentration than the RNP2s in the reaction mixture, and/or blocked nucleic acid molecules comprising at least one bulky modification; and a polymerase and a plurality of nucleotides.

Additionally provided is a composition of matter comprising: ribonucleoprotein complexes (RNPs) comprising a nucleic acid-guided nuclease and a gRNA that is not complementary to the target nucleic acid of interest; wherein the second nucleic acid-guided nuclease optionally comprises a variant nuclease engineered such that single stranded DNA is cleaved faster than double stranded DNA is cleaved, wherein the variant nuclease comprises at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules, and wherein the variant nuclease exhibits both cis- and trans-cleavage activity; a plurality of template molecules comprising sequence homology to the gRNA; and a plurality of the blocked primer molecules comprising a sequence complementary to the template molecules, wherein the blocked primer molecules cannot be extended by a polymerase, and wherein the blocked primer molecules comprise: a first region recognized by the RNP2; one or more second regions not complementary to the first region forming at least one loop; and one or more third regions complementary to and hybridized to the first region forming at least one clamp, wherein the blocked primer molecules optionally each comprise at least one bulky modification, and wherein the plurality of blocked primer molecules and the RNPs optionally are at a concentration where the blocked nucleic acid molecules are at a molar concentration equal to or greater than the molar concentration of the RNPs in the reaction mixture, and wherein the composition comprises at least one of a variant nuclease, a concentration ratio where blocked nucleic acid molecules are at a higher molar concentration than the RNP2s in the reaction mixture, and/or blocked nucleic acid molecules comprising at least one bulky modification; and a polymerase and a plurality of nucleotides.

In some aspects of these embodiments, the reaction mixture further comprises reporter moieties, wherein the reporter moieties produce a detectable signal upon trans-cleavage activity by the RNP2 to identify the presence of one or more nucleic acid targets of interest in the sample. In some aspects, the reporter moieties are not coupled to the blocked primer molecules, and wherein upon cleavage by RNP2, a signal from the reporter moiety is detected; yet in other aspects, the reporter moieties are coupled to the blocked primer molecules, and wherein upon cleavage by RNP2, a signal from the reporter moiety is detected.

In some aspects of all embodiments comprising bulky modifications, the bulky modifications are about 1 nm in size, and in some aspects, the bulky modifications are about 0.9 nm, 0.8 nm, 0.7 nm, 0.6 nm, 0.5 nm, 0.4 nm, 0.3 nm, 0.2 nm, or 0.1 nm in size. In some aspects, the bulky modifications are about 0.9 nm, 0.8 nm, 0.7 nm, 0.6 nm, 0.5 nm, 0.4 nm, 0.3 nm, 0.2 nm, or 0.1 nm in size. In some aspects, the blocked nucleic acid molecules include bulky modifications and wherein there are two bulky modifications with one bulky modification located on the 5′ end of the blocked nucleic acid molecule and one bulky modification located on the 3′ end of the blocked nucleic acid molecule, and where the 5′ and 3′ ends comprising the two bulky modifications are less than 11 nm from one another. In other aspects, the bulky modification is on a 5′ end of blocked nucleic acid molecules and may be selected from the group of 5′ Fam (6-fluorescein amidite); Black Hole Quencher-1-5′; biotin TEG (15 atom triethylene glycol spacer); biotin-5′; and cholesterol TEG (15 atom triethylene glycol spacer). In other aspects, the bulky modification is on a 3′ end of the blocked nucleic acid molecules and may be selected from the group of Black Hole Quencher-1-3′; biotin-3′; and TAMRA-3′ (carboxytetramethylrhodamine). In some aspects, a bulky modification is between two internal nucleic acid residues of the blocked nucleic acid molecules and may be selected from the group of Cy3 internal and Cy5, and in some aspects, the bulky modification is an internal nucleotide base modification and may be selected from the group of biotin deoxythymidine dT; disthiobiotin NHS; and fluorescein dT.

In some aspects of these embodiments, the blocked nucleic acid molecules or blocked primer molecules comprise a structure represented by any one of Formulas I-IV, wherein Formulas I-IV are in the 5′-to-3′ direction: (a)

-   wherein A is 0-15 nucleotides in length; -   B is 4-12 nucleotides in length; -   L is 3-25 nucleotides in length; -   J is an integer between 1 and 10; -   C is 4-15 nucleotides in length; -   M is 1-25 nucleotides in length or is absent, wherein if M is absent     then A-(B-L)J-C and T-D are separate nucleic acid strands; -   T is 17-135 nucleotides in length and comprises at least 50%     sequence complementarity to B and C; and -   D is 0-10 nucleotides in length and comprises at least 50% sequence     complementarity to A;

(b)

-   wherein D is 0-10 nucleotides in length; -   T-T′ is 17-135 nucleotides in length; -   T′ is 1-10 nucleotides in length and does not hybridize with T; -   C is 4-15 nucleotides in length and comprises at least 50% sequence     complementarity to T; -   L is 3-25 nucleotides in length and does not hybridize with T; -   B is 4-12 nucleotides in length and comprises at least 50% sequence     complementarity to T; -   J is an integer between 1 and 10; -   A is 0-15 nucleotides in length and comprises at least 50% sequence     complementarity to D;

(c)

-   wherein T is 17-135 nucleotides in length; -   D is 0-10 nucleotides in length; -   M is 1-25 nucleotides in length or is absent, wherein if M is absent     then T-D and A-(B-L)J-C are separate nucleic acid strands; -   A is 0-15 nucleotides in length and comprises at least 50% sequence     complementarity to D; -   B is 4-12 nucleotides in length and comprises at least 50% sequence     complementarity to T; -   L is 3-25 nucleotides in length; -   J is an integer between 1 and 10; and -   C is 4-15 nucleotides in length; or

(d)

-   wherein T is 17-31 nucleotides in length (e.g., 17-100, 17-50, or     17-25); -   D is 0-15 nucleotides in length; -   M is 1-25 nucleotides in length; -   A is 0-15 nucleotides in length and comprises a sequence     complementary to D; and -   L is 3-25 nucleotides in length; -   p is 0 or 1; -   C is 4-15 nucleotides in length and comprises a sequence     complementary to T.

In some aspects, (a) T of Formula I comprises at least 80% sequence complementarity to B and C; (b ) D of Formula I comprises at least 80% sequence complementarity to A; (c) C of Formula II comprises at least 80% sequence complementarity to T; (d) B of Formula II comprises at least 80% sequence complementarity to T; (e) A of Formula II comprises at least 80% sequence complementarity to D; (f) A of Formula III comprises at least 80% sequence complementarity to D; (g) B of Formular III comprises at least 80% sequence complementarity to T; (h) A of Formula IV comprises at least 80% sequence complementarity to D; and/or (i) C of Formula IV comprises at least 80% sequence complementarity to T.

In some aspects, the variant nucleic acid-guided nuclease is a Type V variant nucleic acid-guided nuclease. In some aspects, the one or both of the RNP1 and the RNP2 comprise a nucleic acid-guided nuclease selected from Cas3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas14, Cas12h, Cas12i, Cas12j, Cas13a, or Cas13b.

In some aspects of the embodiments that comprise a variant nucleic acid-guided nuclease, the variant nucleic acid-guided nuclease comprises at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules wherein the mutation is selected from mutations to amino acid residues K538, Y542 and K595 in relation to SEQ ID NO:1 and equivalent amino acid residues in orthologs. In some embodiments, there are at least two mutations to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules selected from mutations to amino acid residues K538, Y542 and K595 in relation to SEQ ID NO:1 and equivalent amino acid residues in orthologs and in other aspects, there are at least three mutations to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules selected from mutations to amino acid residues K538, Y542 and K595 in relation to SEQ ID NO:1 and equivalent amino acid residues in orthologs. In some aspects, the variant nucleic acid-guided nuclease comprises at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules, wherein the at least one mutation is selected from mutations to amino acid residues K548, N552 and K607 in relation to SEQ ID NO:2; mutations to amino acid residues K534, Y538 and R591 in relation to SEQ ID NO:3; mutations to amino acid residues K541, N545 and K601 in relation to SEQ ID NO:4; mutations to amino acid residues K579, N583 and K635 in relation to SEQ ID NO:5; mutations to amino acid residues K613, N617 and K671 in relation to SEQ ID NO:6; mutations to amino acid residues K613, N617 and K671 in relation to SEQ ID NO:7; mutations to amino acid residues K617, N621 and K678 in relation to SEQ ID NO:8; mutations to amino acid residues K541, N545 and K601 in relation to SEQ ID NO:9; mutations to amino acid residues K569, N573 and K625 in relation to SEQ ID NO:10; mutations to amino acid residues K562, N566 and K619 in relation to SEQ ID NO:11; mutations to amino acid residues K645, N649 and K732 in relation to SEQ ID NO:12; mutations to amino acid residues K548, N552 and K607 in relation to SEQ ID NO:13; mutations to amino acid residues K592, N596 and K653 in relation to SEQ ID NO:14; or mutations to amino acid residues K521, N525 and K577 in relation to SEQ ID NO:15.

In some aspects, the variant nucleic acid-guided nuclease comprises at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules, wherein single stranded DNA is cleaved 1.2 to 2.5 times faster than double stranded DNA is cleaved, at least three to four times faster than double stranded DNA is cleaved, and in some aspects, single stranded DNA is cleaved at least five times faster than double stranded DNA is cleaved. In aspects, the variant nucleic acid-guided nuclease exhibits cis- and trans-cleavage activity.

In some aspects, the variant nucleic acid-guided nuclease comprises at least two mutations to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules, and in some aspects, the variant nuclease comprises at least three mutations to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules.

In any of the embodiments comprising a concentration ratio where blocked nucleic acid molecules are at a higher molar concentration than the RNP2s in the reaction mixture, certain aspects provide that the concentration of the blocked nucleic acid molecules and the RNP2s are at a concentration ratio of at least 1.5 blocked nucleic acid molecules to 1 RNP2 in the reaction mixture, and in some aspects, the concentration of the blocked nucleic acid molecules and the RNP2s are at a concentration ratio of at least 2 blocked nucleic acid molecules to 1 RNP2 in the reaction mixture or at least 3 blocked nucleic acid molecules to 1 RNP2, or at least 3.5 blocked nucleic acid molecules to 1 RNP2, or at least 4 blocked nucleic acid molecules to 1 RNP2, or at least 4.5 blocked nucleic acid molecules to 1 RNP2, or at least 5 blocked nucleic acid molecules to 1 RNP2, or at least 5.5 blocked nucleic acid molecules to 1 RNP2, or at least 6 blocked nucleic acid molecules to 1 RNP2, or at least 6.5 blocked nucleic acid molecules to 1 RNP2, or at least 7.5 blocked nucleic acid molecules to 1 RNP2, or at least 7.5 blocked nucleic acid molecules to 1 RNP2, or at least 8 blocked nucleic acid molecules to 1 RNP2, or at least 8.5 blocked nucleic acid molecules to 1 RNP2, or at least 9 blocked nucleic acid molecules to 1 RNP2, or at least 9.5 blocked nucleic acid molecules to 1 RNP2, or at least 10 blocked nucleic acid molecules to 1 RNP2.

In further embodiments there is provided a variant Cas12a nuclease engineered such that single stranded DNA is cleaved faster than double stranded DNA is cleaved, wherein the variant Cas12a nuclease comprises at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules and wherein the variant Cas12a nuclease exhibits both cis- and trans-cleavage activity. In some aspects, wherein the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K538, Y542 and K595 in relation to SEQ ID NO:1; the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K548, N552 and K607 in relation to SEQ ID NO:2; the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K534, Y538 and R591 in relation to SEQ ID NO:3; the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K541, N545 and K601 in relation to SEQ ID NO:4; the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K579, N583 and K635 in relation to SEQ ID NO:5; the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K613, N617 and K671 in relation to SEQ ID NO:6; the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K613, N617 and K671 in relation to SEQ ID NO:7; the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K617, N621 and K678 in relation to SEQ ID NO:8; the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K541, N545 and K601 in relation to SEQ ID NO:9; the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K569, N573 and K625 in relation to SEQ ID NO:10; the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K562, N566 and K619 in relation to SEQ ID NO:11; the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K645, N649 and K732 in relation to SEQ ID NO:12; the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K548, N552 and K607 in relation to SEQ ID NO:13; the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K592, N596 and K653 in relation to SEQ ID NO:14; or the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K521, N525 and K577 in relation to SEQ ID NO:15 including and equivalent amino acid residues in Cas12a orthologs to these SEQ ID Nos: 1-15.

In some aspects, the variant Cas12a nuclease that has been engineered such that single stranded DNA is cleaved faster than double stranded DNA is cleaved comprises any one of SEQ ID NOs: 16-600.

Alternatively, an embodiment provides a single-strand-specific Cas12a nucleic acid-guided nucleases comprising an LbCas12a (i.e., SEQ ID NO: 1) with an acetylated K595 (K595K^(Ac)) residue; an AsCas12a (i.e., SEQ ID NO: 2) with an acetylated K607 (K607K^(Ac)) residue; a CtCas12a (i.e., SEQ ID NO: 3) with an acetylated R591 (R591R^(Ac)) residue; an EeCas12a (i.e., SEQ ID NO: 4) with an acetylated K601 (K607K^(Ac)) residues; an Mb3Cas12a (i.e., SEQ ID NO: 5) with an acetylated K635 (K635K^(Ac)) residue; an FnCas12a (i.e., SEQ ID NO: 6) with an acetylated K671 (K671K^(Ac)) residue; an FnoCas12a (i.e., SEQ ID NO: 7) with an acetylated N671 (N671K^(Ac)) residue; an FbCas12a (i.e., SEQ ID NO: 8) with an acetylated K678 (K678K^(Ac)) residue; an Lb4Cas12a (i.e., SEQ ID NO: 9) with an acetylated K601 (K601K^(Ac)) residue; an MbCas12a (i.e., SEQ ID NO: 10) with an acetylated K625 (K625K^(Ac)) residue; a Pb2Cas12a (i.e., SEQ ID NO: 11) with an acetylated K619 (K619K^(Ac)) residue; a PgCas12a (i.e., SEQ ID NO: 12) with an acetylated K732 (K732K^(Ac)) residue; an AaCas12a (i.e., SEQ ID NO: 13) with an acetylated K607 (K607K^(Ac)) residue; a BoCas12a (i.e., SEQ ID NO: 14) with an acetylated K653 (K653K^(Ac)) residue; or an CmaCas12a (i.e., SEQ ID NO: 15) with an acetylated K577 (K577K^(Ac)) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be a Cas12a ortholog acetylated at the amino acid of the ortholog equivalent to K595 of SEQ ID NO:1.

These aspects and other features and advantages of the invention are described below in more detail.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings in which:

FIG. 1A is an overview of a prior art quantitative PCR (“qPCR”) assay where target nucleic acids of interest from a sample are amplified before detection.

FIG. 1B is an overview of the general principles underlying the nucleic acid-guided nuclease cascade assay described in detail herein where target nucleic acids of interest from a sample do not need to be amplified before detection.

FIG. 1C is an illustration of the unwinding issue that is mitigated by the modalities described herein.

FIG. 2A is a diagram showing the sequence of steps in an exemplary cascade assay utilizing blocked nucleic acid molecules.

FIG. 2B is a diagram showing an exemplary blocked nucleic acid molecule and a method for unblocking the blocked nucleic acid molecules of the disclosure.

FIG. 2C shows schematics of several exemplary blocked nucleic acid molecules containing the structure of Formula I, as described herein.

FIG. 2D shows schematics of several exemplary blocked nucleic acid molecules containing the structure of Formula II, as described herein.

FIG. 2E shows schematics of several exemplary blocked nucleic acid molecules containing the structure of Formula III, as described herein.

FIG. 2F shows schematics of several exemplary blocked nucleic acid molecules containing the structure of Formula IV, as described herein.

FIG. 2G shows an exemplary single-stranded blocked nucleic acid molecule with a design able to block R-loop formation with an RNP complex, thereby blocking activation of the trans-nuclease activity of an RNP complex (i.e., RNP2).

FIG. 2H shows schematics of exemplary circularized blocked nucleic acid molecules.

FIG. 3A is a diagram showing the sequence of steps in an exemplary cascade assay involving circular blocked primer molecules and linear template molecules.

FIG. 3B is a diagram showing the sequence of steps in an exemplary cascade assay involving circular blocked primer molecules and circular template molecules.

FIG. 4 illustrates three embodiments of reporter moieties.

FIG. 5 is a simplified block diagram of an exemplary method for designing, synthesizing and screening variant nucleic acid-guided nucleases.

FIG. 6A shows the result of protein structure prediction using Rosetta and SWISS modeling of wildtype LbCas12a (Lachnospriaceae bacterium Cas12a).

FIG. 6B shows the result of example mutations on the LbCas12a protein structure prediction using Rosetta and SWISS modeling of LbCas12a and indicating the PAM regions.

FIG. 7 is a simplified diagram of acetylating the K595 amino acid in the wildtype sequence of LbCas12a (K595K^(AC)).

FIG. 8A is an illustration of a blocked nucleic acid molecule with bulky modifications, cleavage thereof, and steric hindrance at the PAM-interacting (PI) domain in a nucleic acid-guided nuclease caused by 5′ and 3′ modifications to a blocked nucleic acid molecule.

FIG. 8B illustrates five exemplary variations of blocked nucleic acid molecules with bulky modifications.

FIGS. 8C, 8D and 8E list exemplary bulky modifications for 5′, 3′, and internal positions in blocked nucleic acid molecules.

FIG. 9 is an illustration of a lateral flow assay that can be used to detect the cleavage and separation of a signal from a reporter moiety.

FIG. 10A depicts Molecule U29 and describes the properties thereof, where MU29 was used to generate the data shown in FIGS. 10B - 10H.

FIG. 11A shows the result of protein structure prediction using Rosetta and SWISS modeling of LbCas 12a comprising the mutation G532A in the wildtype sequence.

FIG. 11B shows the result of protein structure prediction using Rosetta and SWISS modeling of LbCas12a comprising the mutation K538A in the wildtype sequence.

FIG. 11C shows the result of protein structure prediction using Rosetta and SWISS modeling of LbCas 12a comprising the mutation Y542A in the wildtype sequence.

FIG. 11D shows the result of protein structure prediction using Rosetta and SWISS modeling of LbCas 12a comprising the mutation K595A in the wildtype sequence.

FIG. 11E shows the result of protein structure prediction using Rosetta and SWISS modeling of LbCas12a comprising the mutations G532A, K538A, Y5442A and K595A in the wildtype sequence.

FIG. 11F shows the result of protein structure prediction using Rosetta and SWISS modeling of LbCas 12a comprising the mutation K595D in the wildtype sequence.

FIG. 11G shows the result of protein structure prediction using Rosetta and SWISS modeling of LbCas 12a comprising the mutation K595E in the wildtype sequence.

FIG. 11H shows the result of protein structure prediction using Rosetta and SWISS modeling of LbCas12a comprising the mutations K538A, Y542A and K595D in the wildtype sequence.

FIG. 11I shows the result of protein structure prediction using Rosetta and SWISS modeling of LbCas12a comprising the mutations K538A, Y542A and K595E in the wildtype sequence.

FIGS. 12A - 12G are a series of graphs showing the time for detection of dsDNA and ssDNA both with and without PAM sequences for wildtype LbaCas 12a and engineered variants of LbaCas12a.

It should be understood that the drawings are not necessarily to scale, and that like reference numbers refer to like features.

DEFINITIONS

In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the invention. The terms used herein are intended to have the plain and ordinary meaning as understood by those of ordinary skill in the art.

All of the functionalities described in connection with one embodiment of the compositions and/or methods described herein are intended to be applicable to the additional embodiments of the compositions and/or methods except where expressly stated or where the feature or function is incompatible with the additional embodiments. For example, where a given feature or function is expressly described in connection with one embodiment but not expressly mentioned in connection with an alternative embodiment, it should be understood that the feature or function may be deployed, utilized, or implemented in connection with the alternative embodiment unless the feature or function is incompatible with the alternative embodiment.

Note that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” refers to one or more cells, and reference to “a system” includes reference to equivalent steps, methods and devices known to those skilled in the art, and so forth. Additionally, it is to be understood that terms such as “left,” “right,” “top,” “bottom,” “front,” “rear,” “side,” “height,” “length,” “width,” “upper,” “lower,” “interior,” “exterior,” “inner,” “outer” that may be used herein merely describe points of reference and do not necessarily limit embodiments of the present disclosure to any particular orientation or configuration. Furthermore, terms such as “first,” “second,” “third,” etc., merely identify one of a number of portions, components, steps, operations, functions, and/or points of reference as disclosed herein, and likewise do not necessarily limit embodiments of the present disclosure to any particular configuration or orientation.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing devices, formulations and methodologies that may be used in connection with the presently described invention. Conventional methods are used for the procedures described herein, such as those provided in the art, and demonstrated in the Examples and various general references. Unless otherwise stated, nucleic acid sequences described herein are given, when read from left to right, in the 5′ to 3′ direction. Nucleic acid sequences may be provided as DNA, as RNA, or a combination of DNA and RNA (e.g., a chimeric nucleic acid).

Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both limits, ranges excluding either or both of those included limits are also included in the invention.

The term “and/or” where used herein is to be taken as specific disclosure of each of the multiple specified features or components with or without another. Thus, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

As used herein, the term “about,” as applied to one or more values of interest, refers to a value that falls within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of a stated reference value, unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

As used herein, the terms “binding affinity” or “dissociation constant” or “K_(d)” refer to the tendency of a molecule to bind (covalently or non-covalently) to a different molecule. A high K_(d) (which in the context of the present disclosure refers to blocked nucleic acid molecules or blocked primer molecules binding to RNP2) indicates the presence of more unbound molecules, and a low K_(d) (which in the context of the present disclosure refers to unblocked nucleic acid molecules or unblocked primer molecules binding to RNP2) indicates the presence of more bound molecules. In the context of the present disclosure and the binding of blocked or unblocked nucleic acid molecules or blocked or unblocked primer molecules to RNP2, low K_(d) values are in a range from about 100 fM to about 1 aM or lower (e.g., 100 zM) and high K_(d) values are in the range of 100 nM - 100 µM (10 mM) and thus are about 10⁵- to 10¹⁰-fold or higher as compared to low K_(d) values.

As used herein, the terms “binding domain” or “binding site” refer to a region on a protein, DNA, or RNA, to which specific molecules and/or ions (ligands) may form a covalent or non-covalent bond. By way of example, a polynucleotide sequence present on a nucleic acid molecule (e.g., a primer binding domain) may serve as a binding domain for a different nucleic acid molecule (e.g., an unblocked primer nucleic acid molecule). Characteristics of binding sites are chemical specificity, a measure of the types of ligands that will bond, and affinity, which is a measure of the strength of the chemical bond.

As used herein, the term “blocked nucleic acid molecule” refers to nucleic acid molecules that cannot bind to the first or second RNP complex to activate cis- or trans-cleavage. “Unblocked nucleic acid molecule” refers to a formerly blocked nucleic acid molecule that can bind to the second RNP complex (RNP2) to activate trans-cleavage of additional blocked nucleic acid molecules. A “blocked nucleic acid molecule” may be a “blocked primer molecule” in some embodiments of the cascade assay.

The terms “Cas RNA-guided nucleic acid-guided nuclease” or “CRISPR nuclease” or “nucleic acid-guided nuclease” refer to a CRISPR-associated protein that is an RNA-guided nucleic acid-guided nuclease suitable for assembly with a sequence-specific gRNA to form a ribonucleoprotein (RNP) complex.

As used herein, the terms “cis-cleavage”, “cis-nucleic acid-guided nuclease activity”, “cis-mediated nucleic acid-guided nuclease activity”, “cis-nuclease activity”, “cis-mediated nuclease activity”, and variations thereof refer to sequence-specific cleavage of a target nucleic acid of interest, including an unblocked nucleic acid molecule or synthesized activating molecule, by a nucleic acid-guided nuclease in an RNP complex. Cis-cleavage is a single turn-over cleavage event in that only one substrate molecule is cleaved per event.

The term “complementary” as used herein refers to Watson-Crick base pairing between nucleotides and specifically refers to nucleotides hydrogen-bonded to one another with thymine or uracil residues linked to adenine residues by two hydrogen bonds and cytosine and guanine residues linked by three hydrogen bonds. In general, a nucleic acid includes a nucleotide sequence described as having a “percent complementarity” or “percent homology” to a specified second nucleotide sequence. For example, a nucleotide sequence may have 80%, 90%, or 100% complementarity to a specified second nucleotide sequence, indicating that 8 of 10, 9 of 10, or 10 of 10 nucleotides of a sequence are complementary to the specified second nucleotide sequence. For instance, the nucleotide sequence 3′-TCGA-5′ is 100% complementary to the nucleotide sequence 5′-AGCT-3′; and the nucleotide sequence 3′-ATCGAT-5′ is 100% complementary to a region of the nucleotide sequence 5′-GCTAGCTAG-3′.

As used herein, the term “contacting” refers to placement of two moieties in direct physical association, including in solid or liquid form. Contacting can occur in vitro with isolated cells (for example in a tissue culture dish or other vessel) or in samples or in vivo by administering an agent to a subject.

The term “conservative amino acid substitution” refers to the interchangeability in proteins of amino acid residues having similar side chains. For example, a group of amino acids having aliphatic side chains comprises glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains comprises serine and threonine; a group of amino acids having amide containing side chains comprises asparagine and glutamine; a group of amino acids having aromatic side chains comprises phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains comprises lysine, arginine, and histidine; a group of amino acids having acidic side chains comprises glutamate and aspartate; and a group of amino acids having sulfur containing side chains comprises cysteine and methionine. Exemplary conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine-glycine, and asparagine-glutamine.

A “control” is a reference standard of a known value or range of values.

The terms “guide nucleic acid” or “guide RNA” or “gRNA” refer to a polynucleotide comprising 1) a crRNA region or guide sequence capable of hybridizing to the target strand of a target nucleic acid of interest, and 2) a scaffold sequence capable of interacting or complexing with a nucleic acid-guided nuclease. The crRNA region of the gRNA is a customizable component that enables specificity in every nucleic acid-guided nuclease reaction. A gRNA can include any polynucleotide sequence having sufficient complementarity with a target nucleic acid of interest to hybridize with the target nucleic acid of interest and to direct sequence-specific binding of a ribonucleoprotein (RNP) complex containing the gRNA and nucleic acid-guided nuclease to the target nucleic acid. Target nucleic acids of interest may include a protospacer adjacent motif (PAM), and, following gRNA binding, the nucleic acid-guided nuclease induces a double-stranded break either inside or outside the protospacer region on the target nucleic acid of interest, including on an unblocked nucleic acid molecule or synthesized activating molecule. A gRNA may contain a spacer sequence including a plurality of bases complementary to a protospacer sequence in the target nucleic acid. For example, a spacer can contain about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more bases. The gRNA spacer may be 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or more complementary to its corresponding target nucleic acid of interest. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences. A guide RNA may be from about 20 nucleotides to about 300 nucleotides long. Guide RNAs may be produced synthetically or generated from a DNA template.

“Modified” refers to a changed state or structure of a molecule. Molecules may be modified in many ways including chemically, structurally, and functionally. In one embodiment, a nucleic acid molecule (for example, a blocked nucleic acid molecule) may be modified by the introduction of non-natural nucleosides, nucleotides, and/or internucleoside linkages. In another embodiment, a modified protein (e.g., a modified or variant nucleic acid-guided nuclease) may refer to any polypeptide sequence alteration which is different from the wildtype.

The terms “percent sequence identity”, “percent identity”, or “sequence identity” refer to percent (%) sequence identity with respect to a reference polynucleotide or polypeptide sequence following alignment by standard techniques. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, PSI-BLAST, or Megalign software. In some embodiments, the software is MUSCLE (Edgar, Nucleic Acids Res., 32(5):1792-1797 (2004)). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, in embodiments, percent sequence identity values are generated using the sequence comparison computer program BLAST (Altschul, et al., J. Mol. Biol., 215:403-410 (1990)).

As used herein, the terms “preassembled ribonucleoprotein complex”, “ribonucleoprotein complex”, “RNP complex”, or “RNP” refer to a complex containing a guide RNA (gRNA) and a nucleic acid-guided nuclease, where the gRNA is integrated with the nucleic acid-guided nuclease. The gRNA, which includes a sequence complementary to a target nucleic acid of interest, guides the RNP to the target nucleic acid of interest and hybridizes to it. The hybridized target nucleic acid-gRNA units are cleaved by the nucleic acid-guided nuclease. In the cascade assays described herein, a first ribonucleoprotein complex (RNP1) includes a first guide RNA (gRNA) specific to a target nucleic acid of interest, and a first nucleic acid-guided nuclease, such as, for example, cas12a or cas14a for a DNA target nucleic acid, or cas13a for an RNA target nucleic acid. A second ribonucleoprotein complex (RNP2) for signal amplification includes a second guide RNA specific to an unblocked nucleic acid or synthesized activating molecule, and a second nucleic acid-guided nuclease, which may be different from or the same as the first nucleic acid-guided nuclease.

As used herein, the terms “protein” and “polypeptide” are used interchangeably. Proteins may or may not be made up entirely of amino acids.

As used herein, the term “sample” refers to tissues; cells or component parts; body fluids, including but not limited to peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper’s fluid or pre-ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, and umbilical cord blood. “Sample” may also refer to specimens or aliquots from food; agricultural products; pharmaceuticals; cosmetics, nutraceuticals; personal care products; environmental substances such as soil, water (from both natural and treatment sites), air, or sewer samples; industrial sites and products; and chemicals and compounds. A sample further may include a homogenate, lysate or extract. A sample further refers to a medium, such as a nutrient broth or gel, which may contain cellular components, such as proteins or nucleic acid molecules.

The terms “target DNA sequence”, “target sequence”, “target nucleic acid of interest”, “target molecule of interest”, “target nucleic acid”, or “target of interest” refer to any locus that is recognized by a gRNA sequence in vitro or in vivo. The “target strand” of a target nucleic acid of interest is the strand of the double-stranded target nucleic acid that is complementary to a gRNA. The spacer sequence of a gRNA may be 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 98%, 99% or more complementary to the target nucleic acid of interest. Optimal alignment can be determined with the use of any suitable algorithm for aligning sequences. Full complementarity is not necessarily required provided there is sufficient complementarity to cause hybridization and trans-cleavage activation of an RNP complex. A target nucleic acid of interest can include any polynucleotide, such as DNA (ssDNA or dsDNA) or RNA polynucleotides. A target nucleic acid of interest may be located in the nucleus or cytoplasm of a cell such as, for example, within an organelle of a eukaryotic cell, such as a mitochondrion or a chloroplast, or it can be exogenous to a host cell, such as a eukaryotic cell or a prokaryotic cell. The target nucleic acid of interest may be present in a sample, such as a biological or environmental sample, and it can be a viral nucleic acid molecule, a bacterial nucleic acid molecule, a fungal nucleic acid molecule, or a polynucleotide of another organism, such as a coding or a non-coding sequence, and it may include single-stranded or double-stranded DNA molecules, such as a cDNA or genomic DNA, or RNA molecules, such as mRNA, tRNA, and rRNA. The target nucleic acid of interest may be associated with a protospacer adjacent motif (PAM) sequence, which may include a 2-5 base pair sequence adjacent to the protospacer. In some embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more target nucleic acids can be detected by the disclosed method.

As used herein, the terms “trans-cleavage”, “trans-nucleic acid-guided nuclease activity”, “trans-mediated nucleic acid-guided nuclease activity”, “trans-nuclease activity”, “trans-mediated nuclease activity” and variations thereof refer to indiscriminate, non-sequence-specific cleavage of a target nucleic acid molecule by a nucleic acid-guided nuclease (such as by a Cas12, Cas13, and Cas14) which is triggered by binding of N nucleotides of a target nucleic acid molecule to a gRNA and/or by cis- (sequence-specific) cleavage of a target nucleic acid molecule. Trans-cleavage is a “multiple turn-over” event, in that more than one substrate molecule is cleaved after initiation by a single turn-over cis-cleavage event.

Type V CRISPR/Cas nucleic acid-guided nucleases are a subtype of Class 2 CRISPR/Cas effector nucleases such as, but not limited to, engineered Cas12a, Cas12b, Cas12c, C2c4, C2c8, C2c5, C2c10, C2c9, CasX (Cas12e), CasY (Cas12d), Cas 13a nucleases or naturally-occurring proteins, such as a Cas12a isolated from, for example, Francisella tularensis subsp. novicida (Gene ID: 60806594), Candidatus Methanoplasma termitum (Gene ID: 24818655), Candidatus Methanomethylophilus alvus (Gene ID: 15139718), and [Eubacterium] eligens ATCC 27750 (Gene ID: 41356122), and an artificial polypeptide, such as a chimeric protein.

The term “variant” in the context of the present disclosure refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many if not most regions, identical. A variant and reference polypeptide may differ in one or more amino acid residues (e.g., substitutions, additions, and/or deletions). A variant of a polypeptide may be a conservatively modified variant. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code (e.g., a non-natural amino acid). A variant of a polypeptide may be naturally occurring, such as an allelic variant, or it may be a variant that is not known to occur naturally. Variants include modifications —including chemical modifications — to one or more amino acids that do not involve amino acid substitutions, additions or deletions.

As used herein, the terms “variant engineered nucleic acid-guided nuclease” or “variant nucleic acid-guided nuclease” refer to nucleic acid-guided nucleases have been engineered to mutate the PAM interacting domains in the LbCas 12a (Lachnospriaceae bacterium Cas12a), AsCas 12a (Acidaminococcus sp. BV3L6 Cas12a), CtCas12a (Candidatus Methanoplasma termitum Cas12a), EeCas12a (Eubacterium eligens Cas12a), Mb3Cas12a (Moraxella bovoculi Cas12a), FnCas12a (Francisella novicida Cas12a), FnoCas12a (Francisella tularensis subsp. novicida FTG Cas12a), FbCas12a (Flavobacteriales bacterium Cas12a), Lb4Cas12a (Lachnospira eligens Cas12a), MbCas12a (Moraxella bovoculi Cas12a), Pb2Cas12a (Prevotella bryantii Cas12a), PgCas12a (Candidatus Parcubacteria bacterium Cas12a), AaCas12a (Acidaminococcus sp. Cas12a), BoCas12a (Bacteroidetes bacterium Cas12a), CMaCas12a (Candidatus Methanomethylophilus alvus Mx1201 Cas12a), and to-be-discovered equivalent Cas12a nucleic acid-guided nucleases such that double-stranded DNA (dsDNA) substrates bind to the variant nucleic acid-guided nuclease and are cleaved by the variant nucleic acid-guided nuclease at a slower rate than single-stranded DNA (ssDNA) substrates.

A “vector” is any of a variety of nucleic acids that comprise a desired sequence or sequences to be delivered to and/or expressed in a cell. Vectors are typically composed of DNA, although RNA vectors are also available. Vectors include, but are not limited to, plasmids, fosmids, phagemids, virus genomes, synthetic chromosomes, and the like.

DETAILED DESCRIPTION

The present disclosure provides compositions of matter and methods for cascade assays that detect nucleic acids. The cascade assays allow for massive multiplexing, and provide high accuracy, low cost, minimum workflow and results in less than one minute or, in some embodiments, virtually instantaneously, even at ambient temperatures of about 16 - 20° C. or less up to 48° C. The cascade assays described herein comprise first and second ribonucleoprotein complexes and either blocked nucleic acid molecules or blocked primer molecules. The blocked nucleic acid molecules or blocked primer molecules keep the second ribonucleoprotein complexes “locked” unless and until a target nucleic acid of interest activates the first ribonucleoprotein complex. The methods comprise the steps of providing cascade assay components, contacting the cascade assay components with a sample, and detecting a signal that is generated only when a target nucleic acid of interest is present in the sample.

Early and accurate identification of, e.g., infectious agents, microbe contamination, variant nucleic acid sequences that indicate the presence of diseases such as cancer or contamination by heterologous sources is important in order to select correct treatment; identify tainted food, pharmaceuticals, cosmetics and other commercial goods; and to monitor the environment. Nucleic acid-guided nucleases, such as Type V nucleic acid-guided nucleases, can be utilized for the detection of target nucleic acids of interest associated with diseases, food contamination and environmental threats. However, currently available nucleic acid detection such as quantitative PCR (also known as real time PCR or qPCR) or CRISPR-based detection assays such as SHERLOCK™ and DETECTR™ rely on DNA amplification, which requires time and may lead to changes to the relative proportion of nucleic acids, particularly in multiplexed nucleic acid assays. The lack of rapidity for these detection assays is due to the fact that there is a significant lag phase early in the amplification process where fluorescence above background cannot be detected. With qPCR, for example, there is a lag until the cycle threshold or Ct value, which is the number of amplification cycles required for the fluorescent signal to exceed the background level of fluorescence, is achieved and can be quantified.

The present disclosure describes a signal boost cascade assay and improvements thereto that can detect one or more target nucleic acids of interest (e.g., DNA, RNA and/or cDNA) at attamolar (aM) (or lower) limits in less than one minute and in some embodiments virtually instantaneously without the need for amplifying the target nucleic acid(s) of interest, thereby avoiding the drawbacks of multiplex amplification, such as primer-dimerization. As described in detail below, the cascade assays utilize signal boost mechanisms comprising various components including nucleic acid-guided nucleases, guide RNAs (gRNAs) incorporated into ribonucleoprotein complexes (RNP complexes), blocked nucleic acid molecules or blocked primer molecules, reporter moieties, and, in some embodiments, polymerases and template molecules. A particularly advantageous feature of the cascade assay is that, with the exception of the gRNA in RNP1 (i.e., gRNA1), the cascade assay components are essentially identical no matter what target nucleic acid(s) of interest are being detected, and gRNA1 is easily programmable.

The improvements to the signal amplification or signal boost cascade assay described herein result from preventing undesired unwinding of the blocked nucleic acid molecules in the reaction mix by the second ribonucleoprotein complex (RNP2) before the blocked nucleic acid molecules are unblocked via trans-cleavage, leading to increased efficiency, reduced background, and increased signal-to-noise ratio in the cascade assay. Minimizing undesired unwinding serves two purposes. First, preventing undesired unwinding that happens not as a result of unblocking due to trans-cleavage subsequent to cis-cleavage of the target nucleic acid of interest or trans-cleavage of unblocked nucleic acid molecules — but due to other factors — leads to a “leaky” cascade assay system, which in turn leads to non-specific signal generation.

Second, preventing undesired unwinding limits non-specific interactions between the nucleic acid-guided nucleases (here, in the RNP2s) and blocked nucleic acid molecules such that only blocked nucleic acid molecules that become unblocked due to trans-cleavage activity react with the nucleic acid-guided nucleases. This “fidelity” in the cascade assay leads primarily to desired interactions and limits “wasteful” interactions where the nucleic acid-guided nucleases are essentially acting on blocked nucleic acid molecules rather than unblocked nucleic acid molecules. That is, the nucleic acid-guided nucleases are focused on desired interactions which then leads to immediate signal amplification or boost in the cascade assay.

The present disclosure provides three modalities to minimize leakiness leading to minimal false positives or higher background signal. The present disclosure demonstrates that undesired unwinding of the blocked nucleic acid molecules can be lessened substantially by 1) increasing the molar ratio of the concentration of blocked nucleic acid molecules (equivalent to a target nucleic acid molecule for the RNP2) to be equal to or greater than the molar concentration of RNP2 (e.g., the nucleic acid-guided nuclease in RNP2); 2) engineering the nucleic acid-guided nuclease used in RNP2 so as to increase the time it takes the nucleic acid-guided nuclease to recognize double-strand DNA at least two-fold and preferably three-fold or more; and/or 3) engineering the blocked nucleic acid molecules to include bulky modifications (that is, molecules with a size of about 1 nm or less).

The first modality for minimizing undesired unwinding of the blocked nucleic acid molecules (or blocked primer molecules) is to adjust the relative concentrations of the blocked nucleic acid molecules (or blocked primer molecules) and RNP2s such that the molar concentration of the blocked nucleic acid molecules (or blocked primer molecules) is equal to or greater than the molar concentration of RNP2s. Before the present disclosure, the common wisdom in performing CRISPR detection assays was to use a vast excess of nucleic acid-guided nuclease (e.g., RNP complex) to target.

In most detection assays, the quantity of the target nucleic acid of interest is not known (e.g., the detection assay is performed on a sample with an unknown concentration of target); however, in experiments conducted to determine the level of detection of two CRISPR detection assays known in the art, DETECTR™ and SHERLOCK™, the nucleic acid nuclease was present at ng/µL concentrations and the target of interest was present at very low copy numbers or at femtomolar to attamolar concentration. Thus, the present methods and reagent mixtures not only adjust the relative concentrations of the blocked nucleic acid molecules (or blocked primer molecules) and RNP2s such that the molar concentration of the blocked nucleic acid molecules (or blocked primer molecules) is equal to or greater than the molar concentration of RNP2s, but the molar concentration of RNP2s may still exceed the molar concentration of the blocked nucleic acid molecules by a lesser amount, such as where the molar concentration of RNP2s exceeds the molar concentration of blocked nucleic acid molecules (or blocked target molecules) by 100,000x, 50,000x, 25,000x, 10,000x, 5,000x, 1000x, 500x, 100x, 50x, or 10x or less.

For example, Sun, et al. ran side-by-side comparisons of the DETECTR™ and SHERLOCK™ detection assays, using a concentration of 100 ng/µL LbCas12a in the DETECTR™ assay and a concentration of 20 ng/µL LwCas13a in the SHERLOCK™ assay, where the concentration of the target nucleic acid molecules ranged from 0 copies/µL, 0.1 copies/µL, 0.2 copies/µL, 1.0 copy/µL, 2.0 copies/µL, 5.0 copies/µL, 10.0 copies/µL, and so on up to 200.0 copies/µL. (Sun, et al., J. of Translational Medicine, 12:74 (2021).) In addition, Broughton, et al., ran the DETECTR™ assay using a concentration range of 2.5 copies/µL to 1250 copies/µL target nucleic acid molecules to 40 nM LbCas12 (see, Broughton, et al., Nat. Biotech., 38:870-74 (2020)); and Lee, et al., ran the SHERLOCK™ assay using a concentration range of 10 fM to 50 aM target nucleic acid molecules to 150 nM Cas12 (see Lee, et al., PNAS, 117(41):25722-31 (2020). Thus, the ratio of nucleic acid-guided nuclease to blocked nucleic acid molecule (e.g., target for RNP2) described herein is very different from ratios practiced in the art and this ratio has been determined to limit undesired unwinding of the blocked nucleic acid molecules (or blocked primer molecules).

In a second modality, variant nucleic acid-guided nucleases have been engineered to mutate the domains in the variants that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules in, e.g., Type V nucleic acid-guided nucleases such as the LbCas12a (Lachnospriaceae bacterium Cas12a), AsCas 12a (Acidaminococcus sp. BV3L6 Cas12a), CtCas12a (Candidatus Methanoplasma termitum Cas12a), EeCas12a (Eubacterium eligens Cas12a), Mb3Cas12a (Moraxella bovoculi Cas12a), FnCas12a (Francisella novicida Cas12a), FnoCas12a (Francisella tularensis subsp. novicida FTG Cas12a), FbCas12a (Flavobacteriales bacterium Cas12a), Lb4Cas12a (Lachnospira eligens Cas12a), MbCas12a (Moraxella bovoculi Cas12a), Pb2Cas12a (Prevotella bryantii Cas12a), PgCas12a (Candidatus Parcubacteria bacterium Cas12a), AaCas12a (Acidaminococcus sp. Cas12a), BoCas12a (Bacteroidetes bacterium Cas12a), CMaCas12a (Candidatus Methanomethylophilus alvus Mx1201 Cas12a), and other related nucleic acid-guided nucleases (e.g., homologs and orthologs of these nucleic acid-guided nucleases) also limit unwinding. These variant nucleic acid-guided nucleases have been engineered such that double-stranded DNA (dsDNA) substrates bind to and activate to the variant nucleic acid-guided nucleases slowly, but single-stranded DNA (ssDNA) substrates continue to bind and activate the variant nucleic acid-guided nuclease at a high rate. Thus, the variant nucleic acid-guided nucleases effect a “lock” on the RNP complex (here, the RNP2) vis-à-vis double-strand DNA. Locking RNP2 in this way lessens the likelihood of undesired unwinding of the blocked nucleic acid molecules as described in detail herein (see FIG. 1C and the accompanying discussion). Modifying the nucleic acid-guided nucleases to not recognize dsDNA or to recognize dsDNA is contrary to what is desired in other CRISPR-based diagnostic/detection assays.

Finally, another modality for minimizing undesired unwinding of the blocked nucleic acid molecules is to use “bulky modifications” at the 5′ and/or 3′ ends of the blocked nucleic acid molecules and/or at internal nucleic acid bases of the blocked nucleic acid molecules. Doing so creates steric hindrance at the domains of the nucleic acid-guided nuclease in RNP2 that interact with the PAM region or that interact with surrounding sequences on the blocked nucleic acid molecules, disrupting, e.g., PAM recognition in the target strand and preventing displacement of the non-target strand. Using bulky modifications is yet another path to locking RNP2 to double-strand DNA molecules thereby lessening the likelihood of undesired unwinding of the blocked nucleic acid molecules as described in detail herein (again, see FIG. 1C and the accompanying discussion). “Bulky modifications” include molecules with a size of about 1 nm or less.

FIG. 1A provides a simplified diagram demonstrating a prior art method for quantifying target nucleic acids of interest in a sample; namely, the quantitative polymerase chain reaction or qPCR, which to date may be considered the gold standard for quantitative detection assays. The difference between PCR and qPCR is that PCR is a qualitative technique that indicates the presence or absence of a target nucleic acid of interest in a sample, where qPCR allows for quantification of target nucleic acids of interest in a sample. qPCR involves selective amplification and quantitative detection of specific regions of DNA or cDNA (i.e., the target nucleic acid of interest) using oligonucleotide primers that flank the specific region(s) in the target nucleic acid(s) of interest. The primers are used to amplify the specific regions using a polymerase. Like PCR, repeated cycling of the amplification process leads to an exponential increase in the number of copies of the region(s) of interest; however, unlike traditional PCR, the increase is tracked using an intercalating dye or, as shown in FIG. 1A, a sequence-specific probe (e.g., a “Taq-man probe”) the fluorescence of which is detected in real time. RT-qPCR differs from qPCR in that a reverse transcriptase is used to first copy RNA molecules to produce cDNA before the qPCR process commences.

FIG. 1A is an overview of a qPCR assay where target nucleic acids of interest from a sample are amplified before detection. FIG. 1A shows the qPCR method 10, comprising a double-stranded DNA template 12 and a sequence specific Taq-man probe 14 comprising a region complementary to the target nucleic acid of interest 20, a quencher 16, a quenched fluorophore 18 where 22 denotes quenching between the quencher 16 and quenched fluorophore 18. Upon denaturation, the two strands of the double-stranded DNA template 12 separate into complementary single strands 26 and 28. In the next step, primers 24 and 24′ anneal to complementary single strands 26 and 28, as does the sequence-specific Taq-man probe 14 via the region complementary 20 to the complementary strand 26 of the target nucleic acid of interest. Initially the Taq-man probe is annealed to complementary strand 26 of the target region of interest intact; however, primers 24 and 24′ are extended by polymerase 30 but the Taq-man probe is not, due to the absence of a 3′ hydroxy group. Instead, the exonuclease activity of the polymerase “chews up” the Taq-man probe, thereby separating the quencher 16 from the quenched fluorophore 18 resulting in an unquenched or excited-state fluorophore 34. The fluorescence quenching ensures that fluorescence occurs only when target nucleic acids of interest are present and being copied, where the fluorescent signal is proportional to the number of single-strand target nucleic acids being amplified.

As noted above, the downside to the prior art, currently available detection assays such as qPCR, as well as CRISPR-based reaction assays such as SHERLOCK™ and DETECTR™ is that these assays rely on DNA amplification, which, in addition to issues with multiplexing, significantly hinders the ability to perform rapid testing, e.g., in the field. That is, where the present cascade assay works at ambient temperatures, including room temperatures and below, assays that require amplification of the target nucleic acids of interest do not work well at lower temperatures — even those assays utilizing isothermal amplification — due to non-specific binding of the primers and low polymerase activity. Further, primer design is far more challenging. As for the lack of rapidity of detection assays that require amplification of the target nucleic acids of interest, a significant lag phase occurs early in the amplification process where fluorescence above background cannot be detected, particularly in samples with very low copy numbers of the target nucleic acid of interest. And, again, amplification, particularly multiplex amplification, may cause changes to the relative proportion of nucleic acids in samples that, in turn, lead to artifacts or inaccurate results.

FIG. 1B provides a simplified diagram demonstrating a method (100) of a cascade assay. The cascade assay is initiated when the target nucleic acid of interest (104) binds to and activates a first pre-assembled ribonucleoprotein complex (RNP1) (102). A ribonucleoprotein complex comprises a guide RNA (gRNA) and a nucleic acid-guided nuclease, where the gRNA is integrated with the nucleic acid-guided nuclease. The gRNA, which includes a sequence complementary to the target nucleic acid of interest, guides an RNP complex to the target nucleic acid of interest and hybridizes to it. Typically, preassembled RNP complexes are employed in the reaction mix — as opposed to separate nucleic acid-guided nucleases and gRNAs — to facilitate rapid (and in the present cascade assays, virtually instantaneous) detection of the target nucleic acid(s) of interest.

“Activation” of RNP1 refers to activating trans-cleavage activity of the nucleic acid-guided nuclease in RNP1 (106) by binding of the target nucleic acid-guided nuclease to the gRNA of RNP1, initiating cis-cleavage where the target nucleic acid of interest is cleaved by the nucleic acid-guided nuclease. This binding and/or cis-cleavage activity then initiates trans-cleavage activity (i.e., multi-turnover activity) of the nucleic acid-guided nuclease, where trans-cleavage is indiscriminate, leading to non-sequence-specific cutting of nucleic acid molecules by the nucleic acid-guided nuclease of RNP1 (102). This trans-cleavage activity triggers activation of blocked ribonucleoprotein complexes (RNP2s) (108) in various ways, which are described in detail below. Each newly activated RNP2 (110) activates more RNP2 (108 → 110), which in turn cleave reporter moieties (112). The reporter moieties (112) may be a synthetic molecule linked or conjugated to a quencher (114) and a fluorophore (116) such as, for example, a probe with a dye label (e.g., FAM or FITC) on the 5′ end and a quencher on the 3′ end. The quencher (114) and fluorophore (116) can be about 20-30 bases apart (or about 10-11 nm apart) or less for effective quenching via fluorescence resonance energy transfer (FRET). Reporter moieties also are described in greater detail below.

As more RNP2s are activated (108 → 110), more trans-cleavage activity is activated and more reporter moieties are activated (where here, “activated” means unquenched); thus, the binding of the target nucleic acid of interest (104) to RNP1 (102) initiates what becomes a cascade of signal production (120), which increases exponentially; hence, the terms “signal amplification” or “signal boost.” The cascade assay thus comprises a single turnover event that triggers a multi-turnover event that then triggers another multi-turnover event in a “cascade.” As described below in relation to FIG. 4 , the reporter moieties (112) may be provided as molecules that are separate from the other components of the nucleic acid-guided nuclease cascade assay, or the reporter moieties may be covalently or non-covalently linked to the blocked nucleic acid molecules or synthesized activating molecules (i.e., the target molecules for the RNP2).

As described in detail below, the present description presents three modalities for minimizing undesired unwinding of the blocked nucleic acid molecules (or blocked primer molecules), which possess regions of double-strand DNA, where such unwinding can lead to non-specific signal generation and false positives. The modalities are 1) altering the ratio of the nucleic acid-guided nuclease in RNP2 to the blocked nucleic acid molecules in contravention to the common wisdom for CRISPR detection/diagnostic assays; 2) engineering the nucleic acid-guided nuclease used in RNP2 so that recognition of double-stranded DNA occurs more slowly than for single-strand DNA, in contravention to nucleic acid-guided nucleases that are used in other CRISPR-based detection assays; and 3) modifying the 5′ and/or 3′ ends and/or various internal nucleic acid bases of the blocked nucleic acid molecules. One, two or all three of these modalities may be employed in a given assay.

FIG. 1C is an illustration of the effects of unwinding. FIG. 1C shows at left a double-strand blocked nucleic acid molecule comprising a target strand and a non-target strand, where the non-target strand comprises regions (shown as loops) unhybridized to the target strand. Proceeding right at top, cleavage of the loops in the non-target strand by trans-cleavage initiated by RNP1 or RNP2 destabilizes the double-strand blocked nucleic acid molecule; that is, the now short regions of the non-target strand that are hybridized to the target strand become destabilized and dehybridize. As these short regions dehybridize, the target strand is released and can bind to gRNA2 in RNP2, triggering cis-cleavage of the target strand followed by trans-cleavage of additional blocked nucleic acid molecules. This process is the signal boost assay working as designed.

The pathway at the bottom of FIG. 1C illustrates the effect of undesired unwinding; that is, unwinding due not to trans-cleavage as designed but by other unwinding due to recognition of the blocked nucleic acid molecule by gRNA2 and the nucleic acid-guided nuclease in RNP2. As seen in the alternative pathway at bottom of FIG. 1C, R-loop formation between RNP2 and the blocked nucleic acid molecule (or blocked primer molecule) can still occur due to unwinding of the blocked nucleic acid molecule after gRNA2 identifies the PAM. Indeed, this unwinding can occur even in the absence of a PAM. It is an inherent characteristic of the biology of nucleic acid-guided nucleases.

Various components of the cascade assay, descriptions of how the cascade assays work, and the modalities used to minimize undesired unwinding of the blocked nucleic acid molecules (or blocked primer molecules) are described in detail below.

Target Nucleic Acids of Interest

The target nucleic acid of interest may be a DNA, RNA, or cDNA molecule. Target nucleic acids of interest may be isolated from a sample or organism by standard laboratory techniques or may be synthesized by standard laboratory techniques (e.g., RT-PCR). The target nucleic acids of interest are identified in a sample, such as a biological sample from a subject (including non-human animals or plants), items of manufacture, or an environmental sample (e.g., water or soil). Non-limiting examples of biological samples include blood, serum, plasma, saliva, mucus, a nasal swab, a buccal swab, a cell, a cell culture, and tissue. The source of the sample could be any mammal, such as, but not limited to, a human, primate, monkey, cat, dog, mouse, pig, cow, horse, sheep, and bat. Samples may also be obtained from any other source, such as air, water, soil, surfaces, food, beverages, nutraceuticals, clinical sites or products, industrial sites (including food processing sites) and products, plants and grains, cosmetics, personal care products, pharmaceuticals, medical devices, agricultural equipment and sites, and commercial samples.

In some embodiments, the target nucleic acid of interest is from an infectious agent (e.g., a bacteria, protozoan, insect, worm, virus, or fungus) that affects mammals, including humans. As a non-limiting example, the target nucleic acid of interest could be one or more nucleic acid molecules from bacteria, such as Bordetella parapertussis, Bordetella pertussis, Chlamydia pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae, Acinetobacter calcoaceticus-baumannii complex, Bacteroides fragilis, Enterobacter cloacae complex, Escherichia coli, Klebsiella aerogenes, Klebsiella oxytoca, Klebsiella pneumoniae group, Moraxella catarrhalis, Proteus spp., Salmonella enterica, Serratia marcescens, Haemophilus influenzae, Neisseria meningitidis, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Enterococcus faecalis, Enterococcus faecium, Listeria monocytogenes, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus lugdunensis, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Chlamydia tracomatis, Neisseria gonorrhoeae, Syphilis (Treponema pallidum), Ureaplasma urealyticum, Mycoplasma genitalium, and/or Gardnerella vaginalis. Also, as a non-limiting example, the target nucleic acid of interest could be one or more nucleic acid molecules from a virus, such as adenovirus, coronavirus HKU1, coronavirus NL63, coronavirus 229E, coronavirus OC43, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human metapneumovirus, human rhinovirus, enterovirus, influenza A, influenza A/H1, influenza A/H3, influenza A/H1-2009, influenza B, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4, respiratory syncytial virus, herpes simplex virus 1, herpes simplex virus 2, human immunodeficiency virus (HIV), human papillomavirus, hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), and/or human parvovirus B19 (B 19V). Also, as a non-limiting example, the target nucleic acid of interest could be one or more nucleic acid molecules from a fungus, such as Candida albicans, Candida auris, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis, Cryptococcus neoformans, and/or Cryptococcus gattii. As another non-limiting example, the target nucleic acid of interest could be one or more nucleic acid molecules from a protozoan, such as Trichomonas vaginalis. See, e.g., Table 1 for an exemplary list of human pathogens, Table 2 for an exemplary list of human sexually transmissible diseases.

TABLE 1 Human Pathogens Name Category NCBI Taxonomy ID NCBI Sequence ID Number Acinetobacter baumannii Bacteria 470 GCF_008632635.1 Acinetobacter calcoaceticus Bacteria 471 GCF_002055515.1 Acinetobacter calcoaceticus-baumannii complex Bacteria 909768 Not applicable Anaplasma phagocytophilum Bacteria 948 GCF_000439775.1 Bacillus anthracis Bacteria 1392 GCF_000008445.1 Bacteroides fragilis Bacteria 817 GCF_016889925.1 Bartonella henselae Bacteria 38323 GCF_000612965.1 Bordetella parapertussis Bacteria 519 GCF_004008295.1 Bordetella pertussis Bacteria 520 GCF_004008975.1 Borrelia mayonii Bacteria 1674146 GCF_001936295.1 Borrelia miyamotoi Bacteria 47466 GCF_003431845.1 Brucella abortus Bacteria 235 GCF_000054005.1 Brucella melitensis Bacteria 29459 GCF_000007125.1 Brucella suis Bacteria 29461 GCF_000007505.1 Burkholderia mallei Bacteria 13373 GCF_002346025.1 Burkholderia pseudomallei Bacteria 28450 GCF_000756125.1 Campylobacter jejuni Bacteria 197 GCF_000009085.1 Chlamydia pneumoniae Bacteria 83558 GCF_000007205.1 Chlamydia psittaci Bacteria 83554 GCF_000204255.1 Chlamydia Tracomatis Bacteria 813 GCF_000008725.1 Clostridium botulinum Bacteria 1491 GCF_000063585.1 Clostridium perfringens Bacteria 1502 GCF_020138775.1 Coxiella burnetii Bacteria 777 GCF_000007765.2 Ehrlichia chaffeesis Bacteria 945 GCF_000632965.1 Ehrlichia ewingii Bacteria 947 Not available Ehrlichia ruminantium Bacteria 779 GCF_013460375.1 Enterobacter cloacae Bacteria 550 GCF_000770155.1 Enterobacter cloacae complex Bacteria 354276 Not applicable Enterococcus faecalis Bacteria 1351 GCF_000393015.1 Enterococcus faecium Bacteria 1352 GCF_009734005.1 Escherichia coli Bacteria 562 GCF_000008865.2 Francisella tularensis Bacteria 263 GCF_000156415.1 Gardnerella vaginalis Bacteria 2702 GCF_002861965.1 Haemophilus influenzae Bacteria 727 GCF_000931575.1 Klebsiella aerogenes Bacteria 548 GCF_007632255.1 Klebsiella oxytoca Bacteria 571 GCF_003812925.1 Klebsiella pneumoniae Bacteria 573 GCF_000240185.1 Legionella pneumophila Bacteria 446 GCF_001753085.1 Leptospira interrogans Bacteria 173 GCF_002073495.2 Leptospira kirschneri Bacteria 29507 GCF_000243695.2 Leptospira wolffii Bacteria 409998 GCF_004770635.1 Listeria monocytogenes Bacteria 1639 GCF_000196035.1 Moraxella catarrhalis Bacteria 480 GCF_002080125.1 Mycobacterium tuberculosis Bacteria 1773 GCF_000195955.2 Mycoplasma genitalium Bacteria 2097 GCF_000027325.1 Mycoplasma pneumoniae Bacteria 2104 GCF_900660465.1 Neisseria gonorrhoeae Bacteria 485 GCF_013030075.1 Neisseria meningitidis Bacteria 487 GCF_008330805.1 Proteus hauseri Bacteria 183417 GCF_004116975.1 Proteus mirabilis Bacteria 584 GCF_000069965.1 Proteus penneri Bacteria 102862 GCF_022369495.1 Proteus vulgaris Bacteria 585 GCF_000754995.1 Pseudomonas aeruginosa Bacteria 287 GCF_000006765.1 Rickettsia parkeri Bacteria 35792 GCF_005549115.1 GCA_018610945.1 GCF_000965075.1 GCF_000965085.1 GCF_000284195.1 GCF_000965145.1 Rickettsia prowazekii Bacteria 782 GCF_000277165.1 Rickettsia rickettsii Bacteria 783 GCF_000017445.4 Salmonella bongori Bacteria 54736 GCF_000439255.1 Salmonella enterica Bacteria 28901 GCF_000006945.2 Salmonella enterica Bacteria 28901 GCF_000006945.2 Serratia marcescens Bacteria 615 GCF_003516165.1 Shigella boydii Bacteria 621 GCF_001905915.1 Shigella dysenteriae Bacteria 622 GCF_001932995.2 Shigella flexneri Bacteria 623 GCF_000006925.2 Shigella sonnei Bacteria 624 GCF_013374815.1 Staphylococcus auerus Bacteria 1280 GCF_000013425.1 Staphylococcus enterotoxin B Bacteria 1280 U93688.2 Staphylococcus epidermidis Bacteria 1282 GCF_006094375.1 Staphylococcus lugdunensis Bacteria 28035 GCF_001558775.1 Stenotrophomonas maltophilia Bacteria 40324 GCF_900475405.1 Streptococcus agalactiae Bacteria 1311 GCF_001552035.1 Streptococcus pneumoniae Bacteria 1313 GCF_002076835.1 Streptococcus pyogenes Bacteria 1314 GCF_900475035.1 Treponema pallidum Bacteria 160 GCF_000246755.1 Ureaplasma urealvticum Bacteria 2130 GCF_000021265.1 Vibrio parahaemolyticus Bacteria 670 GCF_000196095.1 Vibrio vulnificus Bacteria 672 GCF_002204915.1 Yersinia enterocolitica Bacteria 630 GCF_001160345.1 Yersinia pestis Bacteria 632 GCF_000222975.1 Candida albicans Fungus 5476 GCF_000182965.3 Candida auris Fungus 498019 GCF_002775015.1 Candida glabrata Fungus 5478 GCF_000002545.3 Candida parapsilosis Fungus 5480 GCF_000182765.1 Candida tropicalis Fungus 5482 GCF_000006335.3 Coccidioides immitis Fungus 5501 GCF_000149335.2 Coccidioides posadasii Fungus 199306 GCF_000151335.2 Cokeromyces recurvatus Fungus 90255 GCA_000697235.1 Cryptococcus gattii Fungus 37769 GCF_000185945.1 Cryptococcus neoformans Fungus 5207 GCF_000091045.1 Cunninghamella bertholletiae Fungus 90251 GCA_000697215.1 Encephalitozoon cuniculi Fungus 6035 GCF_000091225.1 Encephalitozoon hellem Fungus 27973 GCF_000277815.2 Encephalitozoon intestinalis Fungus 58839 GCF_000146465.1 Enterocystozoon bieneusi Fungus 31281 GCF_000209485.1 Mortierella wolfii Fungus 90253 GCA_016098105.1 Pichia kudriavzevii Fungus 4909 GCF_003054445.1 Saksenaea vasiformis Fungus 90258 GCA_000697055.1 Syncephalastrum racemosum Fungus 13706 GCA_002105135.1 Trichomonas vaginalis Fungus 5722 GCF_000002825.2 Ricinus communis Plant 3988 GCF_019578655.1 Acanthamoeba castellanii Protozoa 5755 GCF_000313135.1 Babesia divergens Protozoa 32595 GCA_001077455.2 Babesia microti Protozoa 5868 GCF_000691945.2 Balamuthia mandrillaris Protozoa 66527 GCA_001185145.1 Cryptosporidium parvum Protozoa 5807 GCF_000165345.1 Cyclospora cayatanensis Protozoa 88456 GCF_002999335.1 Entamoeba histolytica Protozoa 5759 GCF_000208925.1 Giardia lamblia Protozoa 5741 GCF_000002435.2 Naegleria fowleri Protozoa 5763 GCF_008403515.1 Toxoplasma gondii Protozoa 5811 GCF_000006565.2 Alkhumra hemorrhagic fever virus Virus 172148 JF416961.1 Argentinian mammarenavirus Virus 2169991 GCF_000856545.1 Betacoronavirus 1 Virus 694003 GCF_000862505.1 GCF_003972325.1 Black Creek Canal orthohantavirus Virus 1980460 GCF_002817355.1 California encephalitis orthobunyavirus Virus 1933264 GCF_003972565.1 Chapare mammarenavirus Virus 499556 GCF_000879235.1 Chikungunya virus Virus 37124 GCF_000854045.1 Crimean-Congo hemorrhagic fever orthnairovirus Virus 1980519 GCF_000854165.1 Dabie bandavirus Virus 2748958 GCF_000897355.1 GCF_003087855.1 Deer tick virus Virus 58535 MZ148230 to MZ148271 Dengue virus 1 Virus 11053 GCF_000862125.1 Dengue virus 2 Virus 11060 GCF_000871845.1 Dengue virus 3 Virus 11069 GCF_000866625.1 Dengue virus 4 Virus 11070 GCF_000865065.1 Eastern equine encephalitis virus Virus 11021 GCF_000862705.1 Enterovirus A Virus 138948 GCF_002816655.1 GCF_000861905.1 GCF_001684625.1 Enterovirus B Virus 138949 GCF_002816685.1 GCF_000861325.1 Enterovirus C Virus 138950 GCF_000861165.1 Enterovirus D Virus 138951 GCF_000861205.1 GCF_002816725.1 Guanarito mammarenavirus Virus 45219 GCF_000853765.1 Heartland bandavirus Virus 2747342 GCF_000922255.1 Hendra henipavirus Virus 63330 GCF_000852685.1 Hepacivirus C Virus 11103 GCF_002820805.1 GCF_000861845.1 GCF_000871165.1 GCF_000874285.1 GCF_001712785.1 hepatitis A virus Virus 208726 K02990.1 M14707.1 M20273.1 X75215.1 AB020564.1 hepatitis B virus Virus 10407 GCF_000861825.2 hepatitis C virus Virus 11103 GCF_002820805.1 GCF_000861845.1 GCF_000871165.1 GCF_000874285.1 GCF_000874265.1 GCF_001712785.1 Hepatovirus A Virus 12092 GCF_000860505.1 Human adenovirus A Virus 129875 GCF_000846805.1 Human adenovirus B Virus 108098 GCF_000857885.1 Human adenovirus C Virus 129951 GCF_000858645.1 Human adenovirus D Virus 130310 GCF_000885675.1 Human adenovirus E Virus 130308 GCF_000897015.1 Human adenovirus F Virus 130309 GCF_000846685.1 Human adenovirus G Virus 536079 GCF_000847325.1 Human alphaherpesvirus 1 Virus 10298 GCF_000859985.2 Human alphaherpesvirus 2 Virus 10310 GCF_000858385.2 human betaherpesvirus 6A Virus 32603 GCF_000845685.2 human betaherpesvirus 6B Virus 32604 GCF_000846365.1 Human coronavirus 229E Virus 11137 GCF_001500975.1 GCF_000853505.1 Human coronavirus HKU1 Virus 290028 GCF_000858765.1 Human coronavirus NL63 Virus 277944 GCF_000853865.1 Human coronavirus OC43 Virus 31631 GCF_003972325.1 Human gammaherpesvirus 8 Virus 37296 GCF_000838265.1 Human immunodeficiency virus 1 Virus 11676 GCF_000864765.1 Human immunodeficiency virus 2 Virus 11709 GCF_000856385.1 human metapneumovirus Virus 162145 GCF_002815375.1 human papillomavirus Virus GCF_001274345.1 Human polyomavirus 1 Virus 1891762 GCF_000837865.1 Human polyomavirus 2 Virus 1891763 GCF_000863805.1 human rhinovirus A Virus 147711 GCF_000862245.1 GCF_002816835.1 human rhinovirus B Virus 147712 GCF_000861265.1 GCF_002816855.1 human rhinovirus C Virus 463676 GCF_002816885.1 GCF_000872325.1 Influenza A virus Virus 11320 GCF_001343785.1 GCF_000851145.1 GCF_000866645.1 Influenza B virus Virus 11520 GCF_000820495.2 Influenza C virus Virus 11552 GCF_000856665.10 Influenza D virus Virus 1511084 GCF_002867775.1 Japanese encephalitis virus Virus 11072 GCF_000862145.1 Kyasanur Forest disease virus Virus 33743 GCF_002820625.1 La Crosse orthobunyavirus Virus 2560547 GCF_000850965.1 Lassa virus Virus 11620 GCF_000851705.1 Lujo mammarenavirus Virus 649188 GCF_000885555.1 Lyssavirus australis Virus 90961 GCF_000850325.1 Marburg virus Virus NC_001608.3 Measles morbillivirus Virus 11234 GCF_000854845.1 Middle East respiratory syndrome-related coronavirus Virus 1335626 GCF_002816195.1 GCF_000901155.1 Monongahela hantavirus Virus 2259728 MH539865 MH539866 MH539867 New York hantavirus Virus 44755 U36803.1 U36802.1 U36801.1 U09488.1 Nipah henipavirus Virus 121791 GCF_000863625.1 Norwalk virus Virus 11983 GCF_000864005.1 GCF_008703965.1 GCF_008703985.1 GCF_008704025.1 GCF_010478905.1 GCF_000868425.1 Omsk hemorrhagic fever virus Virus 12542 GCF_000855505.1 parainfluenza virus 1 Virus 12730 GCF_000848705.1 NC_003461 parainfluenza virus 2 Virus X57559.1 AF533010 AF533011 AF533012 parainfluenza virus 3 Virus 11216 GCA_006298365.1 GCA_000850205.1 parainfluenza virus 4 Virus 2560526 NC_021928.1 Paslahepevirus balayani Virus 1678141 GCF_000861105.1 Poliovirus Virus 138950 GCF_000861165.1 Primate erythroparvovirus 1 Virus 1511900 GCF_000839645.1 Rabies lyssavirus Virus 11292 GCF_000859625.1 respiratory syncytial virus Virus 12814 GCF_000856445.1 Rift Valley virus Virus 11588 HE687302 HE687307 Saint Louis encephalitis virus Virus 11080 GCF_000866785.1 Sapporo virus Virus 95342 GCF_000849945.1 GCF_000855765.1 GCF_000854265.1 GCF_001008475.1 GCF_000853825.1 SARS-related coronavirus Virus 694009 GCF_000864885.1 GCF_009858895.2 Severe acute respiratory syndrome coronavirus 1 Virus 2901879 NC_004718.3 Severe acute respiratory syndrome coronavirus 2 Virus 2697049 NC_045512.2 Sin Nombre virus Virus 1980491 GCF_000854765.1 Tick-borne encephalitis virus Virus 11084 GCF_000863125.1 Variola major Virus 12870 not available Variola minor Virus 53258 not available Variola virus Virus 10255 GCF_000859885.1 Venezuelan equine encephalitis virus Virus 11036 GCF_000862105.1 West Nile virus Virus 11082 GCF_000861085.1 GCF_000875385.1 Western equine encephalitis virus Virus 11039 GCF_000850885.1 Yellow fever virus Virus 11089 GCF_000857725.1 Zaire ebolavirus Virus 186538 GCF_000848505.1 Zika virus Virus 64320 GCF_000882815.3 GCF_002366285.1

TABLE 2 Human STD pathogens Name Category NCBI Taxonomy ID NCBI Sequence ID Number Pthirus pubis Animal 121228 MT721740.1 Sarcoptes scabiei Animal 52283 GCA_020844145.1 Chlamydia trachomatis Bacteria 813 GCF_000008725.1 Gardnerella vaginalis Bacteria 2702 GCF_002861965.1 Haemophilus ducreyi Bacteria 730 GCF_001647695.1 Mycoplasma genitalium Bacteria 2097 GCF_000027325.1 Neisseria gonorrhoeae Bacteria 485 GCF_013030075.1 Treponema pallidum Bacteria 160 GCF_000246755.1 Trichomonas vaginalis Protozoa 5722 GCF_000002825.2 Hepacivirus C Virus 11103 GCF_002820805.1 Hepatitis B virus Virus 10407 GCF_000861825.2 Hepatitis delta virus Virus 12475 GCF_000856565.1 Hepatovirus A Virus 12092 GCF_000860505.1 Human alphaherpesvirus 1 Virus 10298 GCF_000859985.2 Human immunodeficiency virus 1 Virus 11676 GCF_000864765.1 Human immunodeficiency virus 2 Virus 11709 GCF_000856385.1 Human papillomavirus Virus 10566 GCF_001274345.1

Additionally, the target nucleic acid of interest may originate in an organism such as a bacterium, virus, fungus or other pest that infects livestock or agricultural crops. Such organisms include avian influenza viruses, mycoplasma and other bovine mastitis pathogens, Clostridium perfringens, Campylobacter sp., Salmonella sp., Pospirivoidae, Avsunvirodiae, Panteoea stewartii, Mycoplasma genitalium, Sprioplasma sp., Pseudomonas solanacearum, Erwinia amylovora, Erwinia carotovora, Pseudomonas syringae, Xanthomonas campestris, Agrobacterium tumefaciens, Spiroplasma citri, Phytophthora infestans, Endothia parasitica, Ceratocysis ulmi, Puccinia graminis, Hemilea vastatrix, Ustilage maydis, Ustilage nuda, Guignardia bidwellii, Uncinula necator, Botrytis cincerea, Plasmopara viticola, or Botryotinis fuckleina. See, e.g., Table 3 for an exemplary list of non-human animal pathogens.

TABLE 3 Animal Pathogens Name Category NCBI Taxonomy ID NCBI Sequence ID Number Acarapis woodi Animal 478375 GCA_023170135.1 Aethina tumida Animal 116153 GCF_001937115.1 Chorioptes bovis Animal 420257 Chrysomya bezziana Animal 69364 Cochliomyia hominivorax Animal 115425 GCA_004302925.1 Echinococcus granulosus Animal 6210 GCF_000524195.1 Echinococcus multilocularis Animal 6211 GCA_000469725.3 Gyrodactylus salaris Animal 37629 GCA_000715275.1 Psoroptes ovis Animal 83912 GCA_002943765.1 Sarcoptes scabiei Animal 52283 GCA_020844145.1 Taenia solium Animal 6204 GCA_001870725.1 Trichinella britovi Animal 45882 GCA_001447585.1 Trichinella nativa Animal 6335 GCA_001447565.1 Trichinella nelsoni Animal 6336 GCA_001447455.1 Trichinella papuae Animal 268474 GCA_001447755.1 Trichinella pseudospiralis Animal 6337 GCA_001447645.1 Trichinella spiralis Animal 6334 GCF_000181795.1 Trichinella zimbabwensis Animal 268475 GCA_001447665.1 Tropilaelaps clareae Animal 208209 Tropilaelaps koenigerum Animal 208208 Tropilaelaps mercedesae Animal 418985 GCA_002081605.1 Tropilaelaps thaii Animal 418986 Varroa destructor Animal 109461 GCF_002443255.1 Varroa jacobsoni Animal 62625 GCF_002532875.1 Varroa rindereri Animal 109259 Varroa underwoodi Animal 109260 Anaplasma centrale Bacteria 769 GCF_000024505.1 Anaplasma marginale Bacteria 770 GCF_000020305.1 Bacillus anthracis Bacteria 1392 GCF_000008445.1 Brucella abortus Bacteria 235 GCF_000054005.1 Brucella melitensis Bacteria 29459 GCF_000007125.1 Brucella ovis Bacteria 236 GCF_000016845.1 Brucella suis Bacteria 29461 GCF_000007505.1 Burkholderia mallei Bacteria 13373 GCF_002346025.1 Burkholderia pseudomallei Bacteria 28450 GCF_000756125.1 Campylobacter fetus Bacteria 196 GCF_000015085.1 Candidatus Xenohaliotis californiensis Bacteria 84677 Candidatus Hepatobacter penaei Bacteria 1274402 GCF_000742475.1 Chlamydia abortus Bacteria 83555 GCF_900416725.2 Chlamydia psittaci Bacteria 83554 GCF_000204255.1 Corynebacterium pseudotuberculosis Bacteria 1719 GCF_001865765.1 Coxiella burnetii Bacteria 777 GCF_000007765.2 Ehrlichia ruminantium Bacteria 779 GCF_013460375.1 Francisella tularensis Bacteria 263 GCF_000156415.1 Melissococcus plutonius Bacteria 33970 GCF_003966875.1 Mycobacterium avium Bacteria 1764 GCF_000696715.1 Mycobacterium tuberculosis Bacteria 1773 GCF_000195955.2 Mycoplasma capricolum Bacteria 2095 GCF_000012765.1 Mycoplasma gallisepticum Bacteria 2096 GCF_000286675.1 Mycoplasma mycoides Bacteria 2102 GCF_000023685.1 Mycoplasma putrefaciens Bacteria 2123 GCF_900476175.1 Mycoplasmopsis agalactiae Bacteria 2110 GCF_009150585.1 Mycoplasmopsis synoviae Bacteria 2109 GCF_013393745.1 Paenibacillus larvae Bacteria 1464 GCF_002951935.1 Pasteurella multocida Bacteria 747 GCF_000006825.1 Salmonella enterica Bacteria 28901 GCF_000006945.2 Streptococcus equi Bacteria 1336 GCF_015689455.1 Taylorella equigenitalis Bacteria 29575 GCF_002288025.1 Vibrio parahaemolyticus Bacteria 670 GCF_000196095.1 Batrachochytrium dendrobatidis Fungi 109871 GCF_000203795.1 Batrachochytrium salamandrivorans Fungi 1357716 GCA_021556675.1 Aphanomyces astaci Oomycota 112090 GCF_000520075.1 Aphanomyces invadans Oomycota 157072 GCF_000520115.1 Babesia bigemina Protozoa 5866 GCF_000981445.1 Babesia bovis Protozoa 5865 GCA_000165395.2 Babesia caballi Protozoa 5871 Bonamia exitiosa Protozoa 362532 Bonamia ostreae Protozoa 126728 Leishmania amazonensis Protozoa 5659 GCA_005317125.1 Leishmania braziliensis Protozoa 5660 GCF_000002845.2 Leishmania donovani Protozoa 5661 GCF_000227135.1 Leishmania infantum Protozoa 5671 GCF_000002875.2 Leishmania major Protozoa 5664 GCF_000002725.2 Leishmania mexicana Protozoa 5665 GCF_000234665.1 Leishmania tropica Protozoa 5666 GCA_014139745.1 Marteilia refringens Protozoa 107386 Perkinsus marinus Protozoa 31276 GCF_000006405.1 Perkinsus olseni Protozoa 32597 GCA_013115135.1 Theileria annulata Protozoa 5874 GCF_000003225.4 Theileria equi Protozoa 5872 GCF_000342415.1 Theileria parva Protozoa 5875 GCF_000165365.1 Tritrichomonas foetus Protozoa 1144522 GCA_001839685.1 Trypanosoma brucei Protozoa 5691 GCF_000002445.2 Trypanosoma congolense Protozoa 5692 GCA_002287245.1 Trypanosoma equiperdum Protozoa 5694 GCA_001457755.2 Trypanosoma evansi Protozoa 5697 GCA_917563935.1 Trypanosoma vivax Protozoa 5699 GCA_021307395.1 African horse sickness virus Virus 40050 GCF_000856125.1 African swine fever virus Virus 10497 GCF_000858485.1 Akabane orthobunyavirus Virus 1933178 GCF_000871205.1 Alcelaphine gammaherpesvirus 1 Virus 35252 GCF_000838825.1 Alphaarterivirus equid Virus 2499620 GCF_000860865.1 Alphacoronavirus 1 Virus 693997 GCF_000856025.1 Ambystoma tigrinum virus Virus 265294 GCF_000841005.1 Avian coronavirus Virus 694014 GCF_012271565.1 Avian influenza virus Virus 11309 Avian metapneumovirus Virus 38525 GCF_002989735.1 Avian orthoavulavirus 1 Virus 2560319 GCF_002834085.1 Avihepatovirus A Virus 691956 GCF_000869945.1 Betaarterivirus suid 1 Virus 2499680 GCF_003971765.1 Bluetongue virus Virus 40051 GCF_000854445.3 Bovine alphaherpesvirus 1 Virus 10320 GCF_008777455.1 Bovine leukemia virus Virus 11901 GCF_000853665.1 Camelpox virus Virus 28873 GCF_000839105.1 Caprine arthritis encephalitis virus Virus 11660 GCF_000857525.1 Crimean-Congo hemorrhagic fever orthonairovirus Virus 1980519 GCF_000854165.1 Cyprinid herpesvirus 3 Virus 180230 GCF_000871465.1 Decapod iridescent virus 1 Virus 2560405 GCF_004788555.1 Decapod penstvldensovirus 1 Virus 1513224 GCF_000844705.1 Deformed wing virus Virus 198112 GCF_000852585.1 Eastern equine encephalitis virus Virus 11021 GCF_000862705.1 Epizootic haematopoietic necrosis virus Virus 100217 GCF_001448375.1 Epizootic hemorrhagic disease virus Virus 40054 GCF_000885335.1 Equid alphaherpesvirus 1 Virus 10326 GCF_000844025.1 Equid alphaherpesvirus 4 Virus 10331 GCF_000846345.1 Equine infectious anemia virus Virus 11665 GCF_000847605.1 Foot-and-mouth disease virus Virus 12110 GCF_002816555.1 Frog virus 3 Virus 10493 GCF_002826565.1 Gallid alphaherpesvirus 1 Virus 10386 GCF_000847005.1 Goatpox virus Virus 186805 GCF_000840165.1 Haliotid herpesvirus 1 Virus 1513231 GCF_000900375.1 Hendra henipavirus Virus 63330 GCF_000852685.1 Infectious bursal disease virus Virus 10995 GCF_000855485.1 Infectious spleen and kidney necrosis virus Virus 180170 GCF_000848865.1 Influenza A virus Virus 11320 GCF_000851145.1 Isavirus salaris Virus 55987 GCF_000854145.2 Japanese encephalitis virus Virus 11072 GCF_000862145.1 Lumpy skin disease virus Virus 59509 GCF_000839805.1 Lyssavirus rabies Virus 11292 GCF_000859625.1 Macrobrachium rosenbergii nodavirus Virus 222557 GCA_000856985.1 Middle East respiratory syndrome-related coronavirus Virus 1335626 GCF_002816195.1 Myxoma virus Virus 10273 GCF_000843685.1 Nairobi sheep disease orthonairovirus Virus 1980526 GCF_002117695.1 Nipah henipavirus Virus 121791 GCF_000863625.1 Norwegian salmonid alphavirus Virus 344701 Novirhabdovirus piscine Virus 1980916 GCF_000856505.1 Novirhabdovirus salmonid Virus 1980917 GCF_000850065.1 Penaeid shrimp infectious myonecrosis virus Virus 282786 GCA_000866305.1 Peste des petits ruminants virus Virus 2593991 GCF_000866445.1 Pestivirus C Virus 2170082 GCF_000864685.1 GCF_003034095.1 Pestivirus A Virus 2170080 GCF_000861245.1 Rabbit hemorrhagic disease virus Virus 11976 GCF_000861285.1 Rift Valley fever phlebovirus Virus 1933187 GCF_000847345.1 Rinderpest morbillivirus Virus 11241 GCF_000856645.1 Severe acute respiratory syndrome-related coronavirus Virus 694009 GCF_000864885.1 Sheeppox virus Virus 10266 GCF_000840205.1 Slow bee paralysis virus Virus 458132 GCF_000887395.1 Sprivirus cvprinus Virus 696863 GCF_000850305.1 Suid alphaherpesvirus 1 Virus 10345 GCF_000843825.1 Swine vesicular disease virus Virus 12075 Taura syndrome virus Virus 142102 GCF_000849385.1 Tilapinevirus tilapiae Virus 2034996 GCF_001630085.1 Venezuelan equine encephalitis virus Virus 11036 GCF_000862105.1 Vesiculovirus indiana Virus 1972577 GCF_000850045.1 Visna-maedi virus Virus 2169971 GCF_000849025.1 West Nile Virus Virus 11082 GCF_000861085.1 Western equine encephalitis virus Virus 11039 GCF_000850885.1 White spot syndrome virus Virus 342409 GCF_000848085.2 Yellow head virus Virus 96029 GCF_003972805.1

In some embodiments, other target nucleic acids of interest may be for non-infectious conditions, e.g., to be used for genotyping, including non-invasive prenatal diagnosis of, e.g, trisomies, other chromosomal abnormalities, and known genetic diseases such as Tay Sachs disease and sickle cell anemia. Other target nucleic acids of interest and samples are described herein, such as human biomarkers for cancer. An exemplary list of human biomarkers is in Table 4. Target nucleic acids of interest may include engineered biologics, including cells such as CAR-T cells, or target nucleic acids of interest from very small or rare samples, where only small volumes are available for testing.

TABLE 4 Human Biomarkers Biomarker Disease Sample NCBI Taxonomy ID NCBI Gene ID Aβ42, amyloid beta-protein Alzheimer disease CSF 9606 351 prion protein Alzheimer disease, prion disease CSF 9606 5621 Vitamin D binding protein multiple sclerosis progression CSF 9606 2638 CXCL13 multiple sclerosis CSF 9606 10563 alpha-synuclein parkinsonian disorders CSF 9606 6622 tau protein parkinsonian disorders CSF 9606 4137 Apo II parkinsonian disorders CSF 9606 336 ceruloplasmin parkinsonian disorders CSF 9606 1356 peroxisome proliferation-activated PD receptor parkinsonian disorders CSF 9606 5467 parkin neurogenerative disorders CSF 9606 5071 PTEN induced putative kinase I neurogenerative disorders CSF 9606 65018 DJ-1 (PARK7) neurogenerative disorders CSF 9606 11315 leucine-rich repeat kinase neurogenerative disorders CSF 9606 120892 secretogranin II bipolar disorder CSF 9606 7857 neurofilament light chain axonal degeneration CSF 9606 4747 IL-12B, CXDL13, IL-8 Intrathecal inflammation CSF 9606 3593, 10563, 3576 ACE2 cardiovascular disease blood 9606 59272 alpha-amylase cardiovascular disease saliva 9606 276 alpha-feto protein pregnancy blood 9606 174 albumin urine diabetes 9606 213 albumin, urea albuminuria urine 9606 213 neutrophil gelatinase-associated lipocalin (NGAL) acute kidney injury urine 9606 3934 IL-18 acute kidney injury urine 9606 3606 liver fatty acid binding protein acute kidney injury urine 9606 2168 Dkk-3 prostate cancer semen 9606 27122 autoantibody to CD25 early diagnosis esophageal squamous cell carcinoma blood 9606 hTERT lung cancer blood 9606 7015 CA125 (MUC16) lung cancer blood 9606 94025 VEGF lung cancer blood 9606 7422 IL-2 lung cancer blood 9606 3558 osteopontin lung cancer blood 9606 6696 BRAF, CCNI, EGRF, FGF19, FRS2, GREB1, and LZTS1 lung cancer saliva 9606 673, 16007, 1956, 9965, 10818, 9687, 11178 human epididymis protein 4 ovarian cancer blood 9606 10406 CA125 ovarian cancer saliva 9606 94025 EMP1 nasopharyngeal carcinoma saliva 9606 13730 IL-8 oral cancer saliva 9606 3576 carcinoembryonic antigen oral or salivary malignant tumors saliva 9606 1048 thioredoxin Spinalcellular carcinoma saliva 9606 7295 AIP (aryl hydrocarbon receptor interacting protein) Acute intermittent porphyria, somatotroph adenoma, prolactin-producing pituitary gland adenoma blood 9606 9049 ALK receptor tyrosine kinase Neuroblastoma susceptibility, large cell lymphoma blood 9606 238 BAP1 (BRCA1 associated protein 1) BAP1-related tumor predisposition, melanoma susceptibility blood 9606 8314 BLM Bloom syndrome blood 9606 641 BRCA1 Breast-ovarian cancer susceptibility, familial breast cancer blood 9606 672 BRCA2 Breast-ovarian cancer susceptibility, familial breast cancer, glioma susceptibility blood 9606 675 CASR (calcium sensing receptor) Epilepsy susceptibility blood 9606 846 CDC73 Hyperparathyroidism 2 with jaw tumors blood 9606 79577 CEBPA Acute myloid leukemia blood 9606 1050 EPCAM Colorectal cancer blood 9606 4072 FH hypercholesterolemia blood 9606 2271 GATA2 Acute myeloid leukemia blood 9606 2642 MITF Melanoma susceptibility blood 9606 4286 MSH2 Lynch syndrome blood 9606 4436 MSH3 Endometrial carcinoma blood 9606 4437 MSH6 Endometrial carcinoma, colorectal cancer blood 9606 2956 NF1 Neurofibromatosis, juvenile myelomonocytic leukemia blood 9606 4763 PDGRA Eosinophilic leukemia, recurrent inflammatory gastrointestinal fibroids blood 9606 5156 PHOX2B Neuroblastoma susceptibility blood 9606 8929 POT1 Melanoma susceptibility, glioma susceptibility blood 9606 25913

The target nucleic acids of interest may be taken from environmental samples. A list of exemplary biosafety pathogens is in Table 5, and an exemplary list of known viruses is in Table 6.

TABLE 5 Exemplary Laboratory Biosafety Parasites and Pathogens Name Category NCBI Taxonomy ID Name Category NCBI Taxonomy ID Acarapis woodi Animal 478375 Streptococcus uberis Bacteria 1349 Aethina tumida Animal 116153 Besnoitia besnoiti Chromista 94643 Alaria americana Animal 2282137 Bonamia exitiosa Chromista 362532 Amblyomma americanum Animal 6943 Bonamia ostreae Chromista 126728 Amblyomma maculatum Animal 34609 Amniculicola longissima Fungus 2566060 Amphimerus pseudofelineus Animal Arthroderma amazonicum Fungus 1592210 Ancylostoma braziliense Animal 369059 Aschersonia hypocreoidea Fungus 370936 Ancylostoma caninum Animal 29170 Aspergillago clavatoflava Fungus 41064 Ancylostoma duodenale Animal 51022 Aspergillus acidohumus Fungus 1904037 Anisakis pegreffii Animal 303229 Aspergillus acidus Fungus 1069201 Anisakis simplex Animal 6269 Aspergillus aculeatinus Fungus 487661 Baylisascaris columnaris Animal 575210 Aspergillus aculeatus Fungus 5053 Baylisascaris melis Animal Aspergillus aeneus Fungus 41754 Baylisascaris procyonis Animal 6259 Aspergillus affinis Fungus 1070780 Bunostomum phlebotomum Animal 577651 Aspergillus alabamensis Fungus 657433 Ceratonova shasta Animal 60662 Aspergillus alliaceus Fungus 209559 Chrysomya bezziana Animal 69364 Aspergillus amazonicus Fungus 710228 Cochliomyia hominivorax Animal 115425 Aspergillus ambiguus Fungus 176160 Dicrocoelium dendriticum Animal 57078 Aspergillus amoenus Fungus 1220191 Diphyllobothrium dendriticum Animal 28845 Aspergillus amyloliquefaciens Fungus 296546 Diphyllobothrium latum Animal 60516 Aspergillus amylovorus Fungus 176161 Echinococcus granulosa Animal Aspergillus angustatus Fungus 2783700 Echinococcus multilocularis Animal 6211 Aspergillus anomalus Fungus 454240 Echinococcus oligarthrus Animal 6212 Aspergillus anthodesmis Fungus 37233 Echinococcus shiquicus Animal 260967 Aspergillus apicalis Fungus 478867 Echinococcus vogeli Animal 6213 Aspergillus appendiculatus Fungus 1140386 Echinostoma cinetorchis Animal 1873862 Aspergillus arachidicola Fungus 656916 Echinostoma hortense Animal 48216 Aspergillus ardalensis Fungus 1458899 Echinostoma liei Animal 48214 Aspergillus arvii Fungus 368784 Echinostoma revolutum Animal 48217 Aspergillus askiburgiensis Fungus 1695225 Fasciola hepatica Animal 6192 Aspergillus asperescens Fungus 176163 Fascioloides magna Animal 394415 Aspergillus assulatus Fungus 1245746 Gyrodactylus salaris Animal 37629 Aspergillus astellatus Fungus 1810904 Ixodes pacificus Animal 29930 Aspergillus aurantiobrunneus Fungus 41725 Ixodes ricinus Animal 34613 Aspergillus aurantiopurpureus Fungus 2663348 Ixodes scapularis Animal 6945 Aspergillus aureolatus Fungus 41755 Metagonimus yokogawai Animal 84529 Aspergillus aureoterreus Fungus 41288 Metorchis conjunctus Animal Aspergillus aureus Fungus 309747 Myxobolus cerebralis Animal 59783 Aspergillus auricomus Fungus 138274 Nanophyetus salmincola Animal 240278 Aspergillus australiensis Fungus 1250384 Necator americanus Animal 51031 Aspergillus austroafricanus Fungus 1220192 Oestrus ovis Animal 123737 Aspergillus avenaceus Fungus 36643 Opisthorchis felineus Animal 147828 Aspergillus awamori Fungus 105351 Opisthorchis viverrini Animal 6198 Aspergillus baarnensis Fungus 2070749 Parafilaria bovicola Animal 2282233 Aspergillus baeticus Fungus 1194636 Paragonimus kellicotti Animal 100269 Aspergillus bahamensis Fungus 522521 Paragonimus miyazakii, Animal 59628 Aspergillus bertholletiae Fungus 1226010 Paragonimus westermani Animal 34504 Aspergillus biplanus Fungus 176164 Psoroptes ovis Animal 83912 Aspergillus bisporus Fungus 41753 Rhipicephalus annulatus Animal 34611 Aspergillus bombycis Fungus 109264 Rhipicephalus sanguineus Animal 34632 Aspergillus botswanensis Fungus 1810893 Sarcoptes scabiei Animal 52283 Candida albicans Fungus 5476 Taenia multiceps Animal 94034 Candida glabrata Fungus 5478 Taenia saginata Animal 6206 Candida krusei Fungus 4909 Taenia solium Animal 6204 Candida parapsilosis Fungus 5480 Toxocara canis Animal 6265 Candida tropicalis Fungus 5482 Toxocara cati Animal 6266 Cryptococcus gattii Fungus 37769 Trichinella spiralis Animal 6334 Cryptococcus neoformans Fungus 5207 Trichuris suis Animal 68888 Epidermophyton floccosum Fungus 34391 Trichuris trichiura Animal 36087 Epidermophyton stockdaleae Fungus 74042 Trichuris vulpis Animal 219738 Fusarium acaciae Fungus Tropilaelaps clareae Animal 208209 Fusarium acaciae-mearnsii Fungus 282272 Tropilaelaps mercedesae Animal 418985 Fusarium acicola Fungus Uncinaria stenocephala Animal 125367 Fusarium acremoniopsis Fungus Varroa destructor Animal 109461 Fusarium acridiorum Fungus Actinobacillus pleuropneumonia Bacteria 715 Fusarium acutatum Fungus 78861 Aeromonas hydrophila Bacteria 644 Fusarium aderholdii Fungus Aeromonas salmonicida Bacteria 645 Fusarium adesmiae Fungus Aliarcobacter butzleri Bacteria 28197 Fusarium aduncisporum Fungus Aliarcobacter cryaerophilus Bacteria 28198 Fusarium aecidii-tussilaginis Fungus Aliarcobacter skirrowii Bacteria 28200 Fusarium aeruginosum Fungus Anaplasma centrale Bacteria 769 Fusarium aethiopicum Fungus 569394 Anaplasma marginale Bacteria 770 Fusarium affine Fungus Anaplasma phagocytophilum Bacteria 948 Fusarium agaricorum Fungus Bacillus anthracis Bacteria 1392 Fusarium ailanthinum Fungus Bacillus cereus Bacteria 1396 Fusarium alabamense Fungus Bartonella henselae Bacteria 38323 Fusarium albedinis Fungus Bibersteinia trehalosi Bacteria 47735 Fusarium albertii Fungus Borrelia burgdorferi Bacteria 139 Fusarium albidoviolaceum Fungus Brucella abortus Bacteria 235 Fusarium albiziae Fungus Brucella canis Bacteria 36855 Fusarium albocarneum Fungus Brucella melitensis Bacteria 29459 Fusarium album Fungus Brucella ovis Bacteria 236 Fusarium aleurinum Fungus Brucella suis Bacteria 29461 Fusarium aleyrodis Fungus Burkholderia mallei Bacteria 13373 Fusarium alkanophilum Fungus Burkholderia pseudomallei Bacteria 28450 Fusarium allescheri Fungus Campylobacter coli Bacteria 195 Fusarium allescherianum Fungus Campylobacter fetus fetus Bacteria 32019 Fusarium allii-sativi Fungus Campylobacter fetus venerealis Bacteria 32020 Trichophyton simii Fungus 63406 Campylobacter jejuni Bacteria 197 Trichophyton soudanense Fungus 69891 Chlamydia caviae Bacteria 83557 Trichophyton tonsurans Fungus 34387 Chlamydia felis Bacteria 83556 Trichophyton verrucosum Fungus 63417 Chlamydia muridarum Bacteria 83560 Trichophyton violaceum Fungus 34388 Chlamydia pecorum Bacteria 85991 Ochroma pyramidale Plant 66662 Chlamydia pneumoniae Bacteria 83558 Babesia bigemina Protozoa 5866 Chlamydia psittaci Bacteria 83554 Babesia bovis Protozoa 5865 Chlamydia suis Bacteria 83559 Babesia divergens Protozoa 32595 Chlamydia trachomatis Bacteria 813 Babesia jakimovi Protozoa Chlamydophilus abortus Bacteria Babesia major Protozoa 127461 Clostridium botulinum Bacteria 1491 Babesia occultans Protozoa 536930 Clostridium difficile Bacteria 1496 Babesia ovata Protozoa 189622 Clostridium perfringens Types A, B, C, and D Bacteria Cryptosporidium parvum Protozoa 5807 Coxiella burnetii Bacteria 777 Eimeria acervulina Protozoa 5801 Cronobacter sakazakii Bacteria 28141 Eimeria brunetti Protozoa 51314 Ehrlichia canis Bacteria 944 Eimeria maxima Protozoa 5804 Ehrlichia chaffeensis Bacteria 945 Eimeria meleagridis Protozoa 1431345 Ehrlichia ewingii Bacteria 947 Eimeria necatrix Protozoa 51315 Ehrlichia ondiri Bacteria Eimeria tenella Protozoa 5802 Ehrlichia ruminantium Bacteria 779 Entamoeba histolytica Protozoa 5759 Escherichia coli Bacteria 562 Giardia duodenalis Protozoa 5741 Klebsiella aerogenes Bacteria 548 Giardia lambia Protozoa Klebsiella granulomatis Bacteria 39824 Histomonas meleagridis Protozoa 135588 Klebsiella grimontii Bacteria 2058152 Ichthyobodo necator Protozoa 155203 Klebsiella huaxiensis Bacteria 2153354 Ichthyophthirius multifiliis Protozoa 5932 Klebsiella kielensis Bacteria 2042302 Isospora burrowsi Protozoa Klebsiella michiganensis Bacteria 1134687 Isospora canis Protozoa 1662860 Klebsiella milletis Bacteria 223378 Isospora felis Protozoa 482539 Klebsiella oxytoca Bacteria 571 Isospora neorivolta Protozoa Klebsiella pneumoniae Bacteria 573 Isospora ohioensis Protozoa 279926 Klebsiella quasipneumoniae Bacteria 1463165 Leishmania braziliensis Protozoa 5660 Klebsiella quasivariicola Bacteria 2026240 Leishmania chagasi Protozoa 44271 Klebsiella senegalensis Bacteria 223379 Leishmania infantum Protozoa 5671 Klebsiella steroids Bacteria 1641362 Marteilia refringens Protozoa 107386 Klebsiella variicola Bacteria 244366 Mikrocytos mackini Protozoa 195010 Proteus mirabilis Bacteria 584 Perkinsus marinus Protozoa 31276 Pseudomonas abietaniphila Bacteria 89065 Perkinsus olensi Protozoa Pseudomonas acephalitica Bacteria 407029 Sarcocystis cruzi Protozoa 5817 Pseudomonas acidophila Bacteria 1912599 Sarcocystis hirsuta Protozoa 61649 Pseudomonas adelgestsugas Bacteria 1302376 Sarcocystis hominis Protozoa 61650 Pseudomonas aeruginosa Bacteria 287 Theileria annulata Protozoa 5874 Pseudomonas aestus Bacteria 1387231 Theileria buffei Protozoa Pseudomonas agarici Bacteria 46677 Theileria lestoquardi Protozoa 77054 Pseudomonas akappageensis Bacteria Theileria luwenshuni Protozoa 540482 Pseudomonas alcaligenes Bacteria 43263 Theileria mutans Protozoa 27991 Pseudomonas alcaliphila Bacteria 101564 Theileria orientalis Protozoa 68886 Pseudomonas alginovora Bacteria 37638 Theileria parva Protozoa 5875 Pseudomonas alkanolytica Bacteria Theileria sergenti Protozoa 5877 Pseudomonas alkylphenolica Bacteria 237609 Theileria uilenbergi Protozoa 507731 Pseudomonas allii Bacteria 2740531 Toxoplasma gondii Protozoa 5811 Pseudomonas alliivorans Bacteria 2810613 Trichomonas fetus Protozoa Pseudomonas allokribbensis Bacteria 2774460 Trichomonas gallinae Protozoa 56777 Pseudomonas alloputida Bacteria 1940621 Trichomonas stableri Protozoa 1440121 Pseudomonas alvandae Bacteria 2842348 Trypanosoma brucei Protozoa 5691 Pseudomonas amygdali Bacteria 47877 Trypanosoma congolense Protozoa 5692 Pseudomonas amyloderamosa Bacteria 32043 Trypanosoma cruzi Protozoa 5693 Pseudomonas anatoliensis Bacteria 2710589 Abras virus Virus 2303487 Pseudomonas andersonii Bacteria 147728 Absettarov virus Virus Pseudomonas anguilliseptica Bacteria 53406 Abu Hammad virus Virus 248058 Pseudomonas antarctica Bacteria 219572 Abu Mina virus Virus 248059 Pseudomonas anuradhapurensis Bacteria 485870 Acado virus Virus Pseudomonas arcuscaelestis Bacteria 2710591 Acara virus Virus 2748201 Pseudomonas argentinensis Bacteria 289370 Achiote virus Virus 2036702 Pseudomonas arsenicoxydans Bacteria 702115 Adana virus Virus 1611877 Pseudomonas asgharzadehiana Bacteria 2842349 Adelaide River virus Virus 31612 Pseudomonas asiatica Bacteria 2219225 Adria virus Virus Pseudomonas asplenii Bacteria 53407 Aedes aegypti densovirus Virus 186156 Pseudomonas asturiensis Bacteria 1190415 Aedes albopictus densovirus Virus 35338 Pseudomonas asuensis Bacteria 1825787 Aedes flavivirus Virus 390845 Pseudomonas atacamensis Bacteria 2565368 Aedes galloisi flavivirus Virus 1046551 Pseudomonas atagonensis Bacteria 2609964 Aedes pseudoscutellaris densovirus Virus Pseudomonas aurantiaca Bacteria 86192 Aedes pseudoscutellaris reovirus Virus 341721 Pseudomonas aureofaciens Bacteria 587851 Aedes vexans Virus 7163 Pseudomonas avellanae Bacteria 46257 African horse sickness virus Virus 40050 Pseudomonas aylmerensis Bacteria 1869229 African swine fever virus Virus 10497 Pseudomonas azadiae Bacteria 2843612 Aguacate virus Virus 1006583 Pseudomonas azerbaij anoccidentalis Bacteria Aino virus Virus 11582 Pseudomonas azerbaij anorientalis Bacteria Akabane virus Virus 70566 Pseudomonas azotifigens Bacteria 291995 Alajuela virus Virus 1552846 Pseudomonas azotoformans Bacteria 47878 Alcelaphine gammaherpesvirus 1 Virus 35252 Pseudomonas baetica Bacteria 674054 Alenquer virus Virus 629726 Pseudomonas balearica Bacteria 74829 Aleutian Mink Disease Virus Pseudomonas baltica Bacteria 2762576 Alfuy virus Virus 44017 Pseudomonas bananamidigenes Bacteria 2843610 Alkhumra hemorrhagic fever virus Virus 172148 Pseudomonas bathycetes Bacteria Allpahuayo mammarenavirus Virus 144752 Pseudomonas batumici Bacteria 226910 Almeirim virus Virus Pseudomonas benzenivorans Bacteria 556533 Almendravirus arboretum Virus 1972683 Pseudomonas bijieensis Bacteria 2681983 Almendravirus cootbay Virus 1972685 Pseudomonas blatchfordae Bacteria 254015 Almpiwar virus Virus 318843 Pseudomonas bohemica Bacteria 2044872 Alocasia macrorrhizos Virus 4456 Pseudomonas borbori Bacteria 289003 Altamira virus Virus Pseudomonas borealis Bacteria 84586 Amaparí virus Virus Pseudomonas botevensis Bacteria 2842352 Ambe virus Virus 1926500 Pseudomonas brassicacearum Bacteria 930166 Amga virus Virus 1511732 Pseudomonas brassicae Bacteria 2708063 Amur/Soochong virus Virus Pseudomonas brenneri Bacteria 129817 Anadyr virus Virus 1642852 Pseudomonas bubulae Bacteria 2316085 Anajatuba virus Virus 379964 Pseudomonas campi Bacteria 2731681 Ananindeua virus Virus 1927813 Pseudomonas canadensis Bacteria 915099 Andasibe virus Virus Pseudomonas canavaninivorans Bacteria 2859001 Andes orthohantavirus Virus 1980456 Pseudomonas cannabina Bacteria 86840 Anhanga virus Virus 904722 Pseudomonas capeferrum Bacteria 1495066 Anhembi virus Virus 273355 Pseudomonas capsici Bacteria 2810614 Anopheles A virus Virus 35307 Pseudomonas caricapapayae Bacteria 46678 Anopheles B virus Virus 35308 Pseudomonas carnis Bacteria 2487355 Anopheles flavivirus Virus 2053814 Pseudomonas caspiana Bacteria 1451454 Anopheles gambiae densovirus Virus 487311 Pseudomonas cavernae Bacteria 2320867 Antequera virus Virus 2748239 Pseudomonas cavernicola Bacteria 2320866 Apoi virus Virus 64280 Pseudomonas cedrina Bacteria 651740 Araguari virus Virus 352236 Pseudomonas cellulosa Bacteria 155077 Aransas Bay virus Virus 1428582 Pseudomonas cerasi Bacteria 1583341 Araraquara virus Virus 139032 Pseudomonas chaetocerotis Bacteria Bluetongue virus Virus 40051 Pseudomonas chengduensis Bacteria 489632 Bobaya virus Virus 2818228 Pseudomonas chloritidismutans Bacteria 203192 Bobia virus Virus Pseudomonas chlororaphis Bacteria 587753 Boracéia virus Virus Pseudomonas cichorii Bacteria 36746 Borna disease virus Virus 12455 Pseudomonas citronellolis Bacteria 53408 Botambi virus Virus Pseudomonas clemancea Bacteria 416340 Boteke virus Virus 864698 Pseudomonas coenobios Bacteria Bouboui virus Virus 64295 Pseudomonas coleopterorum Bacteria 1605838 Bourbon virus Virus 1618189 Pseudomonas composti Bacteria 658457 Bovine ephemeral fever virus Virus 11303 Pseudomonas congelans Bacteria 200452 Bovine Herpes Virus 1 Virus Pseudomonas coronafaciens Bacteria 53409 Bovine leukemia virus Virus 11901 Pseudomonas corrugata Bacteria 47879 Bovine orthopneumovirus Virus 11246 Pseudomonas costantinii Bacteria 168469 Bovine viral diarrhea virus 1 Virus 11099 Pseudomonas cremoricolorata Bacteria 157783 Bowe virus Virus 1400425 Pseudomonas cremoris Bacteria 2724178 Bozo virus Virus 273349 Pseudomonas crudilactis Bacteria 2697028 Cumuto virus Virus 1457166 Pseudomonas cuatrocienegasensis Bacteria 543360 Cupixi mammarenavirus Virus 208899 Pseudomonas cyclaminis Bacteria 2781239 Curionopolis virus Virus 490110 Pseudomonas daroniae Bacteria 2487519 Cyprinid herpesvirus 3 Virus 180230 Pseudomonas deceptionensis Bacteria 882211 Czech Aedes vexans flavivirus virus Virus Pseudomonas defluvii Bacteria 1876757 D′Aguilar virus Virus Pseudomonas delhiensis Bacteria 366289 Dabakala virus Virus Pseudomonas denitrificans Bacteria 43306 Dabieshan virus Virus 1167310 Pseudomonas diazotrophicus Bacteria Dak Nong virus Virus 1238455 Pseudomonas diterpeniphila Bacteria 135830 Dakar bat virus Virus 64282 Pseudomonas donghuensis Bacteria 1163398 Dandenong virus Virus 483046 Pseudomonas dryadis Bacteria 2487520 Dashli virus Virus 1764087 Pseudomonas duriflava Bacteria 459528 Deer tick virus Virus 58535 Pseudomonas edaphica Bacteria 2006980 Dengue virus Virus 12637 Pseudomonas ekonensis Bacteria 2842353 Dengue virus 1 virus Virus Pseudomonas elodea Bacteria 179878 Cumuto virus Virus 1457166 Pseudomonas endophytica Bacteria 1563157 Cupixi mammarenavirus Virus 208899 Pseudomonas entomophila Bacteria 312306 Curionopolis virus Virus 490110 Pseudomonas eucalypticola Bacteria 2599595 Lymphocytic choriomeningitis mammarenavirus Virus 11623 Pseudomonas excibis Bacteria Lyssavirus aravan Virus 211977 Pseudomonas extremaustralis Bacteria 359110 Lyssavirus australis Virus 90961 Pseudomonas extremorientalis Bacteria 169669 Lyssavirus lagos Virus 38766 Pseudomonas fakonensis Bacteria 2842355 Lyssavirus spp. Virus 11286 Pseudomonas farris Bacteria 2841207 Lyssavirus bokeloh Virus 1072176 Pseudomonas farsensis Bacteria 2745492 Lyssavirus caucasicus Virus 249584 Pseudomonas ficuserectae Bacteria 53410 Lyssavirus duvenhage Virus 38767 Pseudomonas fildesensis Bacteria 1674920 Lyssavirus irkut Virus 249583 Pseudomonas flavescens Bacteria 29435 Lyssavirus khujand Virus 237716 Pseudomonas flexibilis Bacteria 706570 Lyssavirus mokola Virus 12538 Pseudomonas floridensis Bacteria 1958950 Lyssavirus rabies Virus 11292 Pseudomonas fluorescens Bacteria 294 Lyssavirus shimoni Virus 746543 Pseudomonas fluvialis Bacteria 1793966 Marisma mosquito virus Virus 1105173 Pseudomonas foliumensis Bacteria 2762593 Marituba virus Virus 292278 Pseudomonas fragi Bacteria 296 Marondera virus Virus 108092 Pseudomonas frederiksbergensis Bacteria 104087 Marrakai virus Virus 108088 Pseudomonas fulgida Bacteria 200453 Massila virus Virus Pseudomonas fulva Bacteria 47880 Matariya virus Virus 1272948 Pseudomonas furukawaii Bacteria 1149133 Matruh virus Virus 1678229 Pseudomonas fuscovaginae Bacteria 50340 Matucare virus Virus 908873 Pseudomonas gelidicola Bacteria 1653853 Mayaro virus Virus 59301 Pseudomonas gessardii Bacteria 78544 Mboke virus Virus 273342 Pseudomonas gingeri Bacteria 117681 Mburo virus Virus 2035534 Pseudomonas glareae Bacteria 1577705 Meaban virus Virus 35279 Pseudomonas glycinae Bacteria 1785145 Medjerda Valley virus Virus 1775957 Pseudomonas gozinkensis Bacteria 2774461 Melao virus Virus 35515 Pseudomonas graminis Bacteria 158627 Méno virus Virus Pseudomonas granadensis Bacteria 1421430 Mercadeo virus Virus 1708574 Pseudomonas gregormendelii Bacteria 1628277 Semliki Forest virus Virus 11033 Pseudomonas grimontii Bacteria 129847 Sena Madureira virus Virus 1272957 Pseudomonas guangdongensis Bacteria 1245526 Seoul virus Virus 1980490 Pseudomonas guariconensis Bacteria 1288410 Sepik virus Virus 44026 Pseudomonas guezennei Bacteria 310348 Serra Do Navio virus Virus 45768 Pseudomonas guguanensis Bacteria 1198456 Serra Norte virus Virus 1000649 Pseudomonas guineae Bacteria 425504 Severe fever with thrombocytopenia syndrome virus Virus 1003835 Pseudomonas guryensis Bacteria 2759165 Shamonda virus Virus 159150 Pseudomonas haemolytica Bacteria 2600065 Shark River virus Virus 2303490 Pseudomonas halodenitrificans Bacteria 53411 Shiant Island virus Virus Pseudomonas halodurans Bacteria 28258 Shokwe virus Virus 273359 Pseudomonas halosaccharolytica Bacteria Shuni virus Virus 159148 Pseudomonas halosensibilis Bacteria Silverwater virus Virus 1564099 Pseudomonas hamedanensis Bacteria 2745504 Simbu orthobunyavirus Virus 35306 Pseudomonas helianthi Bacteria 251654 Sin Nombre virus Virus 1980491 Pseudomonas helleri Bacteria 1608996 Sindbis virus Virus 11034 Pseudomonas helmanticensis Bacteria 1471381 Sixgun City virus Virus Pseudomonas huaxiensis Bacteria 2213017 Skinner Tank virus Virus 481886 Pseudomonas hunanensis Bacteria 1247546 Snowshoe hare virus Virus 11580 Pseudomonas hutmensis Bacteria 2707027 Sokoluk virus Virus 64317 Pseudomonas hydrogenothermophila Bacteria 297 Soldado virus Virus 426791 Pseudomonas hydrogenovora Bacteria 39439 Solwezi virus Virus Pseudomonas hydrolytica Bacteria 2493633 Somone virus Virus Pseudomonas indica Bacteria 137658 Sororoca virus Virus 273354 Pseudomonas indoloxydans Bacteria 404407 Souris virus Virus 2010246 Pseudomonas inefficax Bacteria 2078786 South Bay virus Virus 1526514 Pseudomonas iranensis Bacteria 2745503 South River virus Virus 45769 Pseudomonas iridis Bacteria 2710587 Spanish Culex flavivirus virus Virus Pseudomonas izuensis Bacteria 2684212 Spanish Ochlerotatus flavivirus virus Virus Pseudomonas japonica Bacteria 256466 Spondweni virus Virus 64318 Pseudomonas jessenii Bacteria 77298 Sprivirus cyprinus Virus 696863 Pseudomonas jinanensis Bacteria Sripur virus Virus 1620897 Pseudomonas jinjuensis Bacteria 198616 St. Abbs Head virus Virus Pseudomonas juntendi Bacteria 2666183 St. Croix River virus Virus Pseudomonas kairouanensis Bacteria 2293832 St. Louis encephalitis virus Virus 11080 Pseudomonas karstica Bacteria 1055468 Stanfield virus Virus Pseudomonas kermanshahensis Bacteria 2745482 Stratford virus Virus 44027

TABLE 6 Exemplary list of viruses Name NCBI Taxonomy ID Name NCBI Taxonomy ID Name NCBI Taxonomy ID Aalivirus A 2169685 Enterovirus A 138948 Pseudomonas virus Yua 462590 Aarhusvirus dagda 2732762 Enterovirus B 138949 Pseudoplusia includens virus Aarhusvirus katbat 2732763 Enterovirus C 138950 Pseudotevenvirus RB16 329381 Aarhusvirus luksen 2732764 Enterovirus D 138951 Pseudotevenvirus RB43 115991 Aarhusvirus mysterion 2732765 Enterovirus E 12064 Psimunavirus psiM2 2734265 Abaca bunchy top virus 438782 Enterovirus F 1330520 Psipapillomavirus 1 1177762 Abatino macacapox virus 2734574 Enterovirus G 106966 Psipapillomavirus 2 2170170 Abbeymikolo nvirus abbeymikolon 2734213 Enterovirus H 310907 Psipapillomavirus 3 2170171 Abouovirus abouo 1984774 Enterovirus I 2040663 Psittacid alphaherpesvirus 1 50294 Abouovirus davies 1984775 Enterovirus J 1330521 Psittacine atadenovirus A 2003673 Abutilon golden mosaic virus 1926117 Enterovirus K 2169884 Psittacine aviadenovirus B 2169709 Abutilon mosaic Bolivia virus 932071 Enterovirus L 2169885 Psittacine aviadenovirus C 2734577 Abutilon mosaic Brazil virus 1046572 Entnonagintavirus ENT90 2734061 Psittacinepox virus 2169712 Abutilon mosaic virus 10815 Entoleuca entovirus 2734428 Pteridovirus filicis 2734351 Abutilon yellows virus 169102 Enytus montanus ichnovirus Pteridovirus maydis 2734352 Acadevirus PM116 2733576 Ephemerovirus adelaide 1972589 Pteropodid alphaherpesvirus 1 2560693 Acadevirus Pm5460 2733577 Ephemerovirus berrimah 1972594 Pteropox virus 1873698 Acadevirus PM85 2733574 Ephemerovirus febris 1972593 Pteropus associated gemycircularvirus 1 1985395 Acadevirus PM93 2733575 Ephemerovirus kimberley 1972595 Pteropus associated gemycircularvirus 10 1985404 Acadianvirus acadian 1982901 Ephemerovirus koolpinyah 1972596 Ptyasnivirus 1 2734501 Acadianvirus baee 1982902 Ephemerovirus kotonkan 1972587 Pukovnikvirus pukovnik 540068 Acadianvirus reprobate 1982903 Ephemerovirus obodhiang 1972592 Pulverervirus PFR1 2170091 Acanthamoeb a polyphaga mimivirus 212035 Ephemerovirus yata 1972597 Puma lentivirus 12804 Acanthocystis turfacea chlorella virus 1 322019 Epichloe festucae virus 1 382962 Pumpkin polerovirus 2518373 Acara orthobunyavirus 2170053 Epinotia aporema granulovirus 166056 Pumpkin yellow mosaic virus 1410062 Achimota pararubulavirus 1 2560259 Epiphyas postvittana nucleopolyhed rovirus 70600 Punavirus P1 10678 Achimota pararubulavirus 2 2560260 Epirus cherry virus 544686 Punavirus RCS47 2560452 Achromobacter virus Axp3 2169962 Epizootic haematopoietic necrosis virus 100217 Punavirus SJ46 2560732 Acidianus bottle-shaped virus 437444 Epizootic hemorrhagic disease virus 40054 Punique phlebovirus 2734468 Acidianus filamentous virus 2 300186 Eponavirus epona 2734105 Punta Toro phlebovirus 1933186 Acidianus filamentous virus 3 346881 Epseptimavirus 118970sal2 1982565 Puumala orthohantavirus 1980486 Acidianus filamentous virus 6 346882 Epseptimavirus EPS7 491003 Pyrobaculum filamentous virus 1 1805492 Acidianus filamentous virus 7 346883 Epseptimavirus ev123 2732021 Pyrobaculum spherical virus 270161 Acidianus filamentous virus 8 346884 Epseptimavirus ev329 2732022 Qadamvirus SB28 2733953 Acidianus filamentous virus 9 512792 Epseptimavirus LVR16A 2732023 Qalyub orthonairovirus 1980527 Acidianus rod-shaped virus 1 309181 Epseptimavirus mar003J3 2732019 Qingdaovirus J21 2734135 Acidianus spindle-shaped virus 1 693629 Epseptimavirus S113 2732024 Qingling orthophasmavirus 2560694 Acidianus two-tailed virus 315953 Epseptimavirus S114 2732025 Quail pea mosaic virus Acinetobacter virus 133 279006 Epseptimavirus S116 2732026 Quailpox virus 400570 Acintetobacter virus B2 Epseptimavirus S124 2732027 Quaranjavirus johnstonense 688437 Acintetobacter virus B5 Epseptimavirus S126 2732028 Quaranjavirus quaranfilense 688436 Acionnavirus monteraybay 2734078 Epseptimavirus S132 2732029 Qubevirus durum 39803 Acipenserid herpesvirus 2 2871198 Epseptimavirus S133 2732030 Qubevirus faecium 39804 Aconitum latent virus 101764 Epseptimavirus S147 2732031 Quezon mobatvirus 2501382 Acrobasis zelleri entomopoxvirus Epseptimavirus saus132 2732020 Quhwahvirus kaihaidragon 2283289 Actinidia seed borne latent virus 2560282 Epseptimavirus seafire 2732032 Quhwahvirus ouhwah 2201441 Actinidia virus 1 2024724 Epseptimavirus SH9 2732033 Quhwahvirus paschalis 2182400 Actinidia virus A 1112769 Epseptimavirus STG2 2732034 Rabbit associated gemykroznavirus 1 1985420 Actinidia virus B 1112770 Epseptimavirus stitch 1540099 Rabbit fibroma virus 10271 Actinidia virus X 1331744 Epseptimavirus Sw2 2732035 Rabbit hemorrhagic disease virus 11976 Acute bee paralysis virus 92444 Epsilonarterivirus hemcep 2501964 Rabovirus A 1603962 Adana phlebovirus 2734433 Epsilonarterivirus safriver 2501965 Rabovirus B 2560695 Adeno-associated dependoparvo virus A 1511891 Epsilonarterivirus zamalb 2501966 Rabovirus C 2560696 Adeno-associated dependoparvo virus B 1511892 Epsilonpapillo mavirus 1 40537 Rabovirus D 2560697 Adoxophyes honmai entomopoxvirus 1993630 Epsilonpapillo mavirus 2 2169886 Raccoonpox virus 10256 Adoxophyes honmai nucleopolyhed rovirus 224399 Epsilonpolyo mavirus bovis 1891754 Radish leaf curl virus 435646 Adoxophyes orana granulovirus 170617 Eptesipox virus 1329402 Radish mosaic virus 328061 Aedes aegypti entomopoxvirus Equid alphaherpesvirus 1 10326 Radish yellow edge virus 319460 Aedes aegypti Mosqcopia virus Equid alphaherpesvirus 3 80341 Rafivirus A Aedes pseudoscutella ris reovirus 341721 Equid alphaherpesvirus 4 10331 Rafivirus B 2560699 Aegirvirus SCBP42 2733888 Equid alphaherpesvirus 8 39637 Rafivirus C Aeonium ringspot virus 1962503 Equid alphaherpesvirus 9 55744 Raleighvirus darolandstone 2734266 Aeromonas virus 43 Equid gammaherpes virus 2 12657 Raleighvirus raleigh 2734267 Aeropyrum coil-shaped virus 1157339 Equid gammaherpes virus 5 10371 Ramie mosaic Yunnan virus 1874886 Aeropyrum pernix bacilliform virus 1 700542 Equid gammaherpes virus 7 291612 Ranid herpesvirus 1 85655 Aeropyrum pernix ovoid virus 1 1032474 Equine associated gemycircularvirus 1 1985379 Ranid herpesvirus 2 389214 Aerosvirus AS7 2733365 Equine encephalosis virus 201490 Ranid herpesvirus 3 1987509 Aerosvirus av25AhydR2P P 2733364 Equine foamy virus 109270 Ranunculus leaf distortion virus 341110 Aerosvirus ZPAH7 2733366 Equine infectious anemia virus 11665 Ranunculus mild mosaic virus 341111 Affertcholera mvirus CTXphi 141904 Equine mastadenovirus A 129954 Ranunculus mosaic virus 341112 African cassava mosaic Burkina Faso virus 2560285 Equine mastadenovirus B 129955 Raptor siadenovirus A 691961 African cassava mosaic virus 10817 Equine picobirnavirus 2723956 Raspberry bushy dwarf virus 12451 African eggplant mosaic virus 2056161 Equine rhinitis A virus 47000 Raspberry leaf mottle virus 326941 African horse sickness virus 40050 Equine torovirus 329862 Raspberry ringspot virus 12809 African oil palm ringspot virus 185218 Eracentumvirus era103 1985737 Rat associated gemycircularvirus 1 1985405 African swine fever virus 10497 Eracentumvirus S2 2733579 Rat associated porprismacovirus 1 2170126 Agaricus bisporus alphaendornavirus 1 2734345 Eragrostis curvula streak virus 638358 Rattail cactus necrosis-associated virus 1123754 Agaricus bisporus virus 4 Eragrostis minor streak virus 1030595 Rattus norvegicus polyomavirus 1 1679933 Agatevirus agate 1910935 Eragrostis streak virus 496807 Rauchvirus BPP1 194699 Agatevirus bobb 1910936 Erbovirus A 312185 Raven circovirus 345250 Agatevirus Bp8pC 1910937 Erectites yellow mosaic virus 390443 Ravinvirus N15 40631 Ageratum enation alphasatellite 1260769 Eriborus terebrans ichnovirus Recovirus A 2560702 Ageratum enation virus 188333 Erinnyis ello granulovirus 307444 Red clover associated luteovirus Ageratum latent virus 1386090 Eriocheir sinensis reovirus 273810 Red clover cryptic virus 2 1323524 Ageratum leaf curl Buea betasatellite 912035 Ermolevavirus PGT2 2733903 Red clover mottle virus 12262 Ageratum leaf curl Cameroon betasatellite 635076 Ermolevavirus PhiKT 2733904 Red clover necrotic mosaic virus 12267 Ageratum leaf curl Sichuan virus 2182585 Erskinevirus asesino 2169882 Red clover vein mosaic virus 590403 Ageratum leaf curl virus 333293 Erskinevirus EaH2 2169883 Red deerpox virus Ageratum yellow leaf curl betasatellite 169687 Erysimum latent virus 12152 Redspotted grouper nervous necrosis virus 43763 Ageratum yellow vein alphasatellite 187850 Feline associated cyclovirus 1 1987742 Reginaelenavirus rv3LV2017 2734071 Ageratum yellow vein betasatellite 185750 Feline calicivirus 11978 Rehmannia mosaic virus 425279 Ageratum yellow vein China alphasatellite 1454227 Feline foamy virus 53182 Rehmannia virus 1 2316740 Ageratum yellow vein Hualian virus 437063 Feline immunodeficiency virus 11673 Reptilian ferlavirus 122203 Ageratum yellow vein India alphasatellite 1407058 Feline leukemia virus 11768 Reptilian orthoreovirus 226613 Ageratum yellow vein India betasatellite 2010316 Feline morbillivirus 1170234 Rerduovirus RER2 1982376 Ageratum yellow vein Singapore alphasatellite 915293 Felipivirus A Rerduovirus RGL3 1109716 Ageratum yellow vein 2010317 Felixounavirus Alf5 2560439 Restivirus RSS1 2011075 Sri Lanka betasatellite Ageratum yellow vein Sri Lanka virus 222079 Felixounavirus AYO145A 1965378 Reston ebolavirus 186539 Ageratum yellow vein virus 44560 Felixounavirus BPS15Q2 2560723 Reticuloendotheliosis virus 11636 Aghbyvirus ISAO8 2733367 Felsduovirus 4LV2017 2734062 Reyvirus rey 1983751 Aglaonema bacilliform virus 1512278 Felsduovirus Fels2 194701 Rhesus macaque simian foamy virus 2170199 Agricanvirus deimos 1984777 Felsduovirus RE2010 2734063 Rhinolophus associated gemykibivirus 1 2004965 Agricanvirus desertfox 2560433 Felsduovirus 4LV2017 2734062 Rhinolophus associated gemykibivirus 2 2004966 Agricanvirus Ea3570 1984778 Felsduovirus Fels2 194701 Rhinolophus bat coronavirus HKU2 693998 Agricanvirus ray 1984779 Fernvirus shelly 1921560 Rhinolophus ferrumequinum alphacoronavirus HuB-2013 2501926 Agricanvirus simmy50 1984780 Fernvirus sitara 1921561 Rhinovirus A 147711 Agricanvirus specialG 1984781 Festuca leaf streak cytorhabdovirus Rhinovirus B 147712 Agropyron mosaic virus 41763 Fibralongavirus fv2638A 2734233 Rhinovirus C 463676 Agrotis ipsilon multiple nucleopolyhed rovirus 208013 Fibralongavirus QT1 2734234 Rhizidiomyces virus Agrotis segetum granulovirus 10464 Fibrovirus fs1 70203 Rhizoctonia cerealis alphaendornavirus 1 1408133 Agrotis segetum 1962501 Fibrovirus VGJ 1977140 Rhizoctonia magoulivirus 1 2560704 nucleopolyhed rovirus A Agrotis segetum nucleopolyhed rovirus B 1580580 Ficleduovirus FCL2 2560473 Sabo orthobunyavirus 2560716 Agtrevirus AG3 1987994 Ficleduovirus FCV1 2560474 Saboya virus 64284 Agtrevirus SKML39 2169690 Fig badnavirus 1 1034096 Sacbrood virus 89463 Aguacate phlebovirus 2734434 Fig cryptic virus 882768 Saccharomyces 20S RNA narnavirus 186772 Ahlum waterborne virus Figulus sublaevis entomopoxvirus Saccharum streak virus 683179 Ahphunavirus Ahp1 2733368 Figwort mosaic virus 10649 Saclayvirus Aci011 2734138 Ahphunavirus CF7 2733369 Fiji disease virus 77698 Saclayvirus Aci022 2734139 Ahtivirus sagseatwo 2734079 Finch circovirus 400122 Saclayvirus Aci05 2734137 Aichivirus A 72149 Finkel-Biskis-Jinkins murine sarcoma virus 353765 Saetivirus fs2 1977306 Aichivirus B 194965 Finnlakevirus FLiP 2734591 Saetivirus VFJ 1977307 Aichivirus C 1298633 Fionnbharthvirus fionnbharth 2955891 Saffron latent virus 2070152 Aichivirus D 1897731 Fipivirus A Saguaro cactus virus 52274 Aichivirus E 1986958 Fipvunavirus Fpv4 2560476 Saguinine gammaherpesvirus 1 2169901 Aichivirus F 1986959 Firehammervirus CP21 1190451 Saikungvirus HK633 2169924 Ailurivirus A 2560287 Firehammervirus CP220 722417 Saikungvirus HK75 2169925 Aino orthobunyavirus 2560289 Firehammervirus CPt10 722418 Saimiri sciureus polyomavirus 1 1236410 Air potato ampelovirus 1 2560290 Fischettivirus C1 230871 Saimiriine alphaherpesvirus 1 10353 Akabane orthobunyavir us 1933178 Fishburneviru s brusacoram 1983737 Saimiriine betaherpesvirus 4 1535247 Akhmeta virus 2200830 Flamingopox virus 503979 Saimiriine gammaherpesviru s2 10381 Alajuela orthobunyavir us 1933181 Flammulina velutipes browning virus 568090 Saint Floris phlebovirus Alasvirus muscae 2501934 Flaumdravirus KIL2 2560665 Saint Louis encephalitis virus 11080 Alcelaphine gammaherpes virus 1 35252 Flaumdravirus KIL4 2560666 Saint Valerien virus Alcelaphine gammaherpes virus 2 138184 Fletchervirus CP30A 1980966 Sakhalin orthonairovirus 1980528 Alcube phlebovirus 2734435 Gaiavirus gaia 1982148 Sakobuvirus A 1659771 Alcyoneusviru s K641 2560541 Gaillardia latent virus 1468172 Sal Vieja virus 64301 Alcyoneusviru s RaK2 2560545 Gairo mammarenavi rus 1535802 Salacisavirus pssm2 2734140 Alefpapilloma virus 1 2169692 Gajwadongvir us ECBP5 2733916 Salanga phlebovirus 2734471 Alenquer phlebovirus 2734436 Gajwadongvir us PP99 2733917 Salasvirus phi29 10756 Alexandraviru s AD1 2734080 Galaxyvirus abidatro 2560298 Salchichonvirus LP65 298338 Alexandraviru s alexandra 2734081 Galaxyvirus galaxy 2560303 Salehabad phlebovirus 1933188 Alfalfa betanucleorha bdovirus Galinsoga mosaic virus 60714 Salem salemvirus 2560718 Alfalfa cryptic virus 1 Gallid alphaherpesvir us 1 10386 Salivirus A 1330524 Alfalfa enamovirus 1 1770265 Gamaleyaviru s Sb1 1920761 Salmo aquaparamyxovir us 2749930 Alfalfa leaf curl virus 1306546 Gambievirus bolahunense 2501933 Salmon gillpox virus 2734576 Alfalfa mosaic virus 12321 Gamboa orthobunyavir us 1933270 Saphexavirus VD13 1982380 Alfalfa virus S 1985968 Gammaarterivirus lacdeh 2499678 Sapporo virus 95342 Algerian watermelon mosaic virus 515575 Gammanucleo rhabdovirus maydis 2748968 Sarcochilus virus Y 104393 Allamanda leaf curl virus 452758 Gammapapillo mavirus 1 333926 Sashavirus sasha 2734275 Allamanda leaf mottle distortion virus 1317107 Gammapapillo mavirus 10 1175852 Sasquatchvirus Y3 2734143 Alligatorweed stunting virus Gammapapillo mavirus 11 1513256 Sasvirus BFK20 2560392 Allium cepa amalgavirus 1 2058778 Gayfeather mild mottle virus 578305 Satsuma dwarf virus 47416 Allium cepa amalgavirus 2 2058779 Gecko reptillovirus 2560481 Sauletekiovirus AAS23 2734030 Allium virus X 317027 Gelderlandvirus melville 2560727 Saumarez Reef virus 40012 Allpahuayo mammarenavirus 144752 Gelderlandvirus s16 1913658 Saundersvirus Tp84 2170234 Almendravirus almendras 1972686 Gelderlandvirus stml198 1913657 Sauropus leaf curl virus 1130981 Almendravirus arboretum 1972683 Gelderlandvirus stp4a 2560734 Sawgrhavirus connecticut 2734397 Almendravirus balsa 1972684 Gentian mosaic virus 182452 Sawgrhavirus longisland 2734398 Almendravirus chico 1972687 Gentian ovary ringspot virus 1920772 Sawgrhavirus minto 2734399 Almendravirus cootbay 1972685 Geotrupes sylvaticus entomopoxvirus Sawgrhavirus sawgrass 2734400 Almendravirus menghai 2734366 Gequatrovirus G4 1986034 Scale drop disease virus 1697349 Bat associated cyclovirus 6 1987731 Gequatrovirus ID52 1910968 Scallion mosaic virus 157018 Bat associated cyclovirus 7 1987732 Gequatrovirus talmos 1910969 Scapularis ixovirus 2734431 Bat associated cyclovirus 8 1987733 Gerygone associated gemycircularvirus 1 1985381 Scapunavirus scap1 2560792 Bat associated cyclovirus 9 1987734 Gerygone associated gemycircularvirus 2 1985382 Scheffersomyces segobiensis virus L 1300323 Bat coronavirus CDPHE15 1913643 Harrisina brillians granulovirus 115813 Schefflera ringspot virus 2169729 Bat coronavirus HKU10 1244203 Harrisonvirus harrison 1982221 Schiekvirus EFDG1 2560422 Bat Hp-betacoronavirus Zhejiang2013 2501961 Harvey murine sarcoma virus 11807 Schiekvirus EFP01 2734044 Bat mastadenovirus A 1146877 Hautrevirus hau3 1982895 Schiekvirus EfV12 2734045 Bat mastadenovirus B 1146874 Havel River virus 254711 Schistocerca gregaria entomopoxvirus Bat mastadenovirus C 2015370 Hawkeyevirus hawkeye 2169910 Saphexavirus VD13 1982380 Bat mastadenovirus D 2015372 Hazara orthonairovirus 1980522 Sophora yellow stunt alphasatellite 5 2169837 Bat mastadenovirus E 2015374 Heartland bandavirus 2747342 Sorex araneus coronavirus T14 2734504 Bat mastadenovirus F 2015375 Hebius tobanivirus 1 Sorex araneus polyomavirus 1 2560769 Bat mastadenovirus G 2015376 Hedgehog coronavirus 1 1965093 Sorex coronatus polyomavirus 1 2560770 Bat mastadenovirus H Hedwigvirus hedwig 2560502 Sorex minutus polyomavirus 1 2560771 Bat mastadenovirus I Hedyotis uncinella yellow mosaic virus 1428190 Sorghum chlorotic spot virus 107804 Bat mastadenovirus J Hedyotis yellow mosaic betasatellite 1428189 Sorghum mosaic virus 32619 Batai orthobunyavirus 2560341 Heilongjiangvirus Lb 2734110 Sororoca orthobunyavirus 2560772 Batama orthobunyavirus 1933177 Helenium virus S 12171 Sortsnevirus IME279 2734190 Batfish actinovirus 2560342 Helianthus annuus alphaendornavirus 2184469 Sortsnevirus sortsne 2734189 Bavaria virus 2560343 Helicobasidium mompa alphaendornav irus 1 675833 Sosuga pararubulavirus 2560773 Baxtervirus baxterfox 2169730 Helicobasidium mompa partitivirus V70 344866 Soupsvirus soups 1982563 Baxtervirus yeezy 2169731 Helicobasidium mompa totivirus 1-17 196690 Soupsvirus strosahl 2560510 Baylorvirus bv1127AP1 2734055 Helicoverpa armigera granulovirus 489830 Soupsvirus wait 2560513 Baylorvirus PHL101 376820 Helicoverpa armigera nucleopolyhed rovirus 51313 Souris mammarenavirus 2169997 Bayou orthohantavirus 1980459 Helicoverpa armigera stunt virus 37206 Sourvirus sour 2560509 Bcepfunavirus bcepF1 417280 Heliothis armigera entomopoxvirus 10290 South African cassava mosaic virus 63723 Bcepmuvirus bcepMu 264729 Heliothis virescens ascovirus 3a 113366 Southern bean mosaic virus 12139 Bcepmuvirus E255 431894 Heliothis zea nudivirus 29250 Southern cowpea mosaic virus 196398 Bdellomicrovirus MH2K 1986027 Helleborus mosaic virus 592207 Southern elephant seal virus 1159195 Bdellovibrio virus MAC1 Helleborus net necrosis virus 592206 Southern rice black-streaked dwarf virus 519497 Beak and feather disease virus 77856 Helminthospo rium victoriae virus 145S 2560520 Southern tomato virus 591166 Bean calico mosaic virus 31602 Helminthospo rium victoriae virus 190S 45237 Sowbane mosaic virus 378833 Bean chlorosis virus 1227354 Helsettvirus fPS53 2733626 Soybean associated gemycircularvirus 1 1985413 Bean common mosaic necrosis virus 43240 Helsettvirus fPS54ocr 2733628 Sophora yellow stunt alphasatellite 5 2169837 Bean common mosaic virus 12196 Helsettvirus fPS59 2733627 Sorex araneus coronavirus T14 2734504 Bean dwarf mosaic virus 10838 Helsettvirus fPS9 2733625 Sorex araneus polyomavirus 1 2560769 Bean golden mosaic virus 10839 Helsingorvirus Cba121 1918193 Sorex coronatus polyomavirus 1 2560770 Bean golden yellow mosaic virus 220340 Helsingorvirus Cba171 1918194 Sorex minutus polyomavirus 1 2560771 Bean leaf crumple virus 2004460 Jujube mosaic-associated virus 2020956 Sorghum chlorotic spot virus 107804 Bean leafroll virus 12041 Jun jeilongvirus 2560536 Sorghum mosaic virus 32619 Bean mild mosaic virus Juncopox virus Sororoca orthobunyavirus 2560772 Bean necrotic mosaic orthotospovirus 2560344 Jutiapa virus 64299 Sortsnevirus IME279 2734190 Bean pod mottle virus 12260 Jwalphavirus jwalpha 2169963 Switchgrass mosaic-associated virus 2049938 Bean rugose mosaic virus 128790 Kabuto mountain uukuvirus 2747382 Symapivirus A Bean white chlorosis mosaic virus 2169732 Kadam virus 64310 Synechococcus virus SRIM12-08 2734100 Bean yellow disorder virus 267970 Kadipiro virus 104580 Synedrella leaf curl alphasatellite 1544378 Bean yellow mosaic Mexico virus 714310 Kaeng Khoi orthobunyavirus 1933275 Synedrella yellow vein clearing virus 1914900 Bean yellow mosaic virus 12197 Kafavirus SWcelC56 2733923 Synetaeris tenuifemur ichnovirus Bear Canyon mammarenavirus 192848 Kafunavirus KF1 1982588 Syngnathid ichthamaparvovirus 1 2734305 Beauveria bassiana polymycovirus 1 1740646 Kagunavirus golestan 2560464 Synodus synodonvirus 2749934 Beauveria bassiana victorivirus 1 1685109 Kagunavirus K1G 1911008 Tabernariusvirus tabernarius 2560691 Bebaru virus 59305 Kagunavirus K1H 1911010 Tacaiuma orthobunyavirus 611707 Beecentumtre virus B103 10778 Kagunavirus K1ind1 1911007 Tacaribe mammarenavirus 11631 Beet black scorch virus 196375 Kagunavirus K1ind2 1911009 Tacheng uukuvirus 2734606 Beet chlorosis virus 131082 Kagunavirus RP180 2734197 Tahyna orthobunyavirus 2560796 Beet cryptic virus 1 509923 Merremia mosaic virus 77813 Tangaroavirus tv951510a 2733962 Beet cryptic virus 2 912029 Mesta yellow vein mosaic alphasatellite 1705093 Tankvirus tank 1982567 Beet cryptic virus 3 29257 Mesta yellow vein mosaic Bahraich virus 508748 Tapara phlebovirus 2734474 Beet curly top Iran virus 391228 Metamorphoo virus fireman 2734253 Tapirape pacuvirus 2560798 Beet curly top virus 10840 Metamorphoo virus metamorphoo 2734254 Tapwovirus cesti 2509383 Beet mild yellowing virus 156690 Metamorphoo virus robsfeet 2734255 Taranisvirus taranis 2734146 Beet mosaic virus 114921 Metrivirus ME3 2560269 Taro bacilliform CH virus 1634914 Beet necrotic yellow vein virus 31721 Mguuvirus JG068 2733593 Taro bacilliform virus 178354 Beet pseudoyellows virus 72750 Microbacterium virus MuffinTheCat [2] Tarumizu coltivirus 2734340 Beet ringspot virus 191547 Microcystis virus Ma-LMM01 340435 Tataguine orthobunyavirus 2560799 Beet soil-borne mosaic virus 76343 Microhyla letovirus 1 Taterapox virus 28871 Beet soil-borne virus 46436 Micromonas pusilla reovirus 338781 Taupapillomavirus 1 1176148 Beet virus Q 71972 Micromonas pusilla virus SP1 373996 Taupapillomavirus 2 1513274 Beet western yellows virus 12042 Microplitis croceipes bracovirus Taupapillomavirus 3 1961786 Beet yellow stunt virus 35290 Microtus arvalis polyomavirus 1 2006148 Taupapillomavirus 4 2170222 Beet yellows virus 12161 Mukerjeevirus mv52B1 2734186 Taura syndrome virus 142102 Beetle mivirus Mulberry badnavirus 1 1227557 Tawavirus JSF7 2733965 Beetrevirus B3 2560656 Mulberry mosaic dwarf associated virus 1631303 Tea plant necrotic ring blotch virus 2419939 Beetrevirus JBD67 2560663 Mulberry mosaic leaf roll associated virus 1527441 Tefnutvirus siom18 2734147 Beetrevirus JD18 2560664 Mulberry ringspot virus Tegunavirus rlrt 1921705 Beetrevirus PM105 2560675 Mulberry vein banding associated orthotospovirus Tegunavirus yenmtg1 1921706 Beihai picobirnavirus Mule deerpox virus 304399 Tehran phlebovirus 2734475 Beilong jeilongvirus 2560345 Mume virus A 2137858 Telfairia golden mosaic virus 2169737 Bell pepper alphaendornavirus 354328 Mumps orthorubulavirus 2560602 Telfairia mosaic virus 1859135 Bell pepper mottle virus 368735 Mungbean yellow mosaic betasatellite 2010322 Tellina virus 359995 Belladonna mottle virus 12149 Mukerjeevirus mv52B1 2734186 Tellina virus 1 321302 Bellamyvirus bellamy 2734095 Mulberry badnavirus 1 1227557 Telosma mosaic virus 400394 Bellavista orthobunyavirus 2560346 Mulberry mosaic dwarf associated virus 1631303 Tembusu virus 64293 Bellflower vein chlorosis virus 1720595 Mycobacterium virus Tweety 1993864 Tensaw orthobunyavirus 2560800 Bellflower veinal mottle virus 1982660 Mycobacterium virus Wee 1993860 Tent-making bat hepatitis B virus 1508712 Beluga whale coronavirus SW1 694015 Mycobacterium virus Wildcat 1993859 Teseptimavirus YpsPG 2733885 Bendigovirus GMA6 2560495 Mycoreovirus 1 311228 Testudine orthoreovirus Benedictvirus cuco 1071502 Mycoreovirus 2 404237 Testudinid alphaherpesvirus 3 2560801 Benedictvirus tiger 1993876 Mycoreovirus 3 311229 Tete orthobunyavirus 35319 Benevides orthobunyavirus 2170054 Mylasvirus persius 1914020 Tetterwort vein chlorosis virus 1712389 Bequatrovirus avesobmore 1984785 Mynahpox virus 2169711 Teviot pararubulavirus 2560803 Bequatrovirus B4 1918005 Myodes coronavirus 2JL14 Thailand orthohantavirus 1980492 Bequatrovirus bigbertha 1918006 Myodes glareolus polyomavirus 1 2006147 Thalassavirus thalassa 2060093 Bequatrovirus riley 1918007 Myodes jeilongvirus 2560609 Thaumasvirus stim4 2734148 Bequatrovirus spock 1918008 Myodes narmovirus 2560610 Thermoproteus tenax spherical virus 1 292639 Bequatrovirus troll 1918009 Myohalovirus phiH 1980944 Thermoproteus tenax virus 1 10479 Berhavirus beihaiense 2509379 Noxifervirus noxifer 2560671 Thermus virus IN93 1714273 Berhavirus radialis 2509380 Ntaya virus 64292 Thermus virus P23-77 1714272 Berhavirus sipunculi 2509381 Ntepes phlebovirus 2734464 Thetaarterivirus kafuba 2501999 Berisnavirus 1 2734518 Nuarterivirus guemel Thetaarterivirus mikelba 1 2502000 Cacao yellow mosaic virus 12150 Nudaurelia capensis beta virus 85652 Thetapapillomavirus 1 197772 Cacao yellow vein banding virus 2169726 Nudaurelia capensis omega virus 12541 Thetapolyomavirus censtriata 1891755 Cache Valley orthobunyavirus 2560364 Nupapillomavirus 1 334205 Thetapolyomavirus trebernacchii 2218588 Cachoeira Porteira orthobunyavirus 2560365 Nyando orthobunyavirus 1933306 Thetapolyomavirus trepennellii 2170103 Cacipacore virus 64305 Nyavirus midwayense 644609 Thetisvirus ssm1 2734149 Cactus mild mottle virus 229030 Nyavirus nyamaniniense 644610 Thiafora orthonairovirus 1980529 Cactus virus 2 Nyavirus sierranevadaense 1985708 Thimiri orthobunyavirus 1819305 Cactus virus X 112227 Nyceiraevirus nyceirae 2560506 Thin paspalum asymptomatic virus 1352511 Cadicivirus A 1330068 Nyctalus velutinus alphacoronavirus SC-2013 2501928 Thistle mottle virus Cadicivirus B 2560366 Nylanderia fulva virus 1 1871153 Thogotovirus dhoriense 11318 Caenorhabditis elegans Cer1 virus Nymphadoravirus kita 2170041 Thogotovirus thogotoense 11569 Caenorhabditis elegans Cer13 virus Nymphadoravirus nymphadora 2560507 Thomixvirus OH3 2560804 Caeruleovirus Bc431 1985175 Nymphadoravirus zirinka 2170042 Thornevirus SP15 2560336 Caeruleovirus Bcp1 1985176 Oat blue dwarf virus 56879 Thosea asigna virus 83810 Caeruleovirus BCP82 1985177 Oat chlorotic stunt virus 146762 Thottopalayam thottimvirus 2501370 Caeruleovirus BM15 1985178 Oat dwarf virus 497863 Thunberg fritillary mosaic virus 299200 Caeruleovirus deepblue 1985179 Oat golden stripe virus 45103 Thysanoplusia orichalcea nucleopolyhedro virus 101850 Caeruleovirus JBP901 1985180 Oxbow orthohantavirus 1980484 Tiamatvirus PSSP7 268748 Cafeteria roenbergensis virus 1513235 Oxyplax ochracea nucleopolyhed rovirus 2083176 Tibetan frog hepatitis B virus 2169919 Cafeteriavirus -dependent mavirus 1932923 Paadamvirus RHEph01 2733939 Tibrovirus alphaekpoma 1987018 Caimito pacuvirus 2734421 Pacific coast uukuvirus Tibrovirus beatrice 2170224 Cajanus cajan Panzee virus Pacui pacuvirus 2560617 Tibrovirus betaekpoma 1987019 Caladenia virus A 1198147 Paenibacillus virus Willow Tibrovirus coastal 1972586 Calanthe mild mosaic virus 73840 Pagavirus S05C849 2733940 Tibrovirus congo 1987017 Cali mammarenavirus 2169993 Pagevirus page 1921185 Tibrovirus sweetwater 1987013 Calibrachoa mottle virus 204928 Pagevirus palmer 1921186 Tibrovirus tibrogargan 1972584 California encephalitis orthobunyavirus 1933264 Pagevirus pascal 1921187 Tick associated circovirus 1 2560805 California reptarenavirus 2170175 Pagevirus pony 1921188 Tick associated circovirus 2 2560806 Caligid hexartovirus Pagevirus pookie 1921189 Tick-borne encephalitis virus 11084 Caligrhavirus caligus 2560367 Pagoda yellow mosaic 1505530 Tico phebovirus 2734476 associated virus Caligrhavirus lepeophtheirus 2560551 Paguroni virus 1 2508237 Tidunavirus pTD1 2560834 Caligrhavirus salmonlouse 2560736 Pahexavirus ATCC29399B C 1982252 Tidunavirus VP4B 2560833 Calla lily chlorotic spot orthotospovirus 2560368 Pahexavirus pirate 1982303 Tiger puffer nervous necrosis virus 43764 Calla lily latent virus 243560 Pahexavirus procrass1 1982304 Tigray orthohantavirus 2560807 Callistephus mottle virus 1886606 Pahexavirus SKKY 1982305 Tigrvirus E122 431892 Callitrichine gammaherpes virus 3 106331 Pahexavirus solid 1982306 Tigrvirus E202 431893 Calopogonium yellow vein virus Pahexavirus stormborn 1982307 Tobacco leaf curl Comoros virus 439423 Camel associated drosmacovirus 1 2169876 Pahexavirus wizzo 1982308 Tobacco leaf curl Cuba virus 336987 Camel associated drosmacovirus 2 2169877 Pahsextavirus pAh6C 2733975 Tobacco leaf curl Dominican Republic virus 2528965 Camel associated porprismacovirus 1 2170105 Pairvirus Lo5R7ANS 2733941 Tobacco leaf curl Japan betasatellite 2010326 Camel associated porprismacovirus 2 2170106 Pakpunavirus CAb02 1921409 Tobacco leaf curl Patna betasatellite 2010327 Camel associated porprismacovirus 3 2170107 Pahexavirus pirate 1982303 Tobacco leaf curl Pusa virus 905054 Camel associated porprismacovirus 4 2170108 Pahexavirus procrass1 1982304 Tobacco leaf curl Thailand virus 409287 Camelpox virus 28873 Pahexavirus SKKY 1982305 Tobacco leaf curl Yunnan virus 211866 Campana phlebovirus 2734442 Pea necrotic yellow dwarf virus 753670 Tobacco leaf curl Zimbabwe virus 223337 Campoletis aprilis ichnovirus Pea seed-borne mosaic virus 12208 Tobacco leaf rugose virus 196691 Campoletis flavicincta ichnovirus Pea stem necrosis virus 199361 Veracruzvirus heldan 1032892 Camptochiron omus tentans entomopoxvirus Pea streak virus 157777 Veracruzvirus rockstar 2003502 Campylobacter virus IBB35 1006972 Pea yellow stunt virus 1436892 Verbena latent virus 134374 Camvirus amela 1982882 Peach chlorotic mottle virus 471498 Verbena virus Y 515446 Camvirus CAM 1982883 Peach latent mosaic viroid 12894 Vernonia crinkle virus 1925153 Canary circovirus 142661 Peach marafivirus D 2169999 Vernonia yellow vein betasatellite 666635 Canarypox virus 44088 Peach mosaic virus 183585 Vernonia yellow vein Fujian alphasatellite 2169908 Candida albicans Tca2 virus Peach rosette mosaic virus 65068 Vernonia yellow vein Fujian betasatellite 2050589 Candida albicans Tca5 virus Peanut chlorotic streak virus 35593 Vernonia yellow vein Fujian virus 1001341 Candiru phlebovirus 1933182 Peanut clump virus 28355 Vernonia yellow vein virus 367061 Canid alphaherpesvirus 1 170325 Peanut yellow mosaic virus Versovirus VfO3K6 2011076 Canine associated gemygorvirus 1 1985425 Pear blister canker viroid 12783 Verticillium dahliae chrysovirus 1 759389 Canine circovirus 1194757 Peaton orthobunyavirus 2560627 Vesicular exanthema of swine virus 35612 Canine mastadenovirus A 10537 Peatvirus peat2 2560629 Vesiculovirus alagoas 1972579 Canine morbillivirus 11232 Pecan mosaic-associated virus 1856031 Vesiculovirus bogdanovac 1972567 Canna yellow mottle associated virus 2560371 Pecentumvirus A511 40523 Whitefly-associated begomovirus 7 2169744 Canna yellow mottle virus 419782 Penicillum brevicompactum polymycovirus 1 2734569 White-tufted-ear marmoset simian foamy virus 2170205 Canna yellow streak virus 433462 Pennisetum mosaic virus 221262 Whitewater Arroyo mammarenavirus 46919 Cannabis cryptic virus 1115692 Pepino mosaic virus[3] Wifcevirus ECML117 2734154 Cano Delgadito orthohantavirus 1980463 Pepo aphid-borne yellows virus 1462681 Wifcevirus FEC19 2734155 Canoe virus canoe 2734056 Pepper chat fruit viroid 574040 Wifcevirus WFC 2734156 Cao Bang orthohantavirus 1980464 Pepper chlorotic spot orthotospovirus 2734493 Wifcevirus WFH 2734157 Caper latent virus 1031708 Phietavirus X2 320850 Wigeon coronavirus HKU20 1159908 Capim orthobunyavirus 1933265 Phifelvirus FL1 1633149 Wild cucumber mosaic virus 70824 Capistrivirus KSF1 2011077 Phikmvvirus 15pyo 2733349 Wild melon banding virus Capraria yellow spot virus 2049955 Phlox virus S 436066 Wild onion symptomless virus 1862127 Caprine alphaherpesvirus 1 39944 Phnom Penh bat virus 64894 Wild potato mosaic virus 187977 Caprine arthritis encephalitis virus 11660 Phocid alphaherpesvirus 1 47418 Wild tomato mosaic virus 400396 Caprine gammaherpes virus 2 135102 Phocid gammaherpes virus 2 47419 Wild Vitis latent virus 2560839 Caprine respirovirus 3 2560372 Phocid gammaherpes virus 3 2560643 Wilnyevirus billnye 2560486 Capsicum chlorosis orthotospovirus 2560373 Phocine morbillivirus 11240 Wilsonroadvirus Sd1 2734007 Capsicum India alphasatellite 2734586 Pholetesor ornigis bracovirus Winged bean alphaendornavirus 1 2169693 Captovirus AFV1 235266 Phthorimaea operculella granulovirus 192584 Winklervirus chi14 2560752 Capuchin monkey hepatitis B virus 2163996 Phutvirus PPpW4 2733655 Wiseana signata nucleopolyhedro virus 65124 Caraparu orthobunyavirus 1933290 Phyllosphere sclerotimonavi rus Wissadula golden mosaic virus 51673 Carbovirus queenslandense 2136037 Physalis mottle virus 72539 Wissadula yellow mosaic virus 1904884 Dyonupapillo mavirus 1 1513250 Physarum polycephalum Tp1 virus Wisteria badnavirus 1 1973265 Dyoomegapap illomavirus 1 1918731 Phytophthora alphaendornavirus 1 310750 Wisteria vein mosaic virus 201862 Dyoomikronp apillomavirus 1 1513251 Picardvirus picard 2734264 Witwatersrand orthobunyavirus 2560841 Dyophipapillo mavirus 1 1920493 Pidgey pidchovirus 2509390 Wizardvirus twister6 2170253 Dyopipapillo mavirus 1 1513252 Piedvirus IMEDE1 2733947 Wizardvirus wizard 2170254 Dyopsipapillo mavirus 1 1920498 Pienvirus R801 2733373 Woesvirus woes 1982751 Dyorhopapillo mavirus 1 1513253 Pifdecavirus IBBPF7A 2733657 Wolkberg orthobunyavirus 2170059 Dyosigmapapi llomavirus 1 1513254 Plum bark necrosis stem pitting- 675077 Wongorr virus 47465 associated virus Dyotaupapillo mavirus 1 1932910 Plum pox virus 12211 Wongtaivirus HK542 2169922 Dyothetapapill omavirus 1 1235662 Plumeria mosaic virus 1501716 Woodchuck hepatitis virus 35269 Dyoupsilonpa pillomavirus 1 1932912 Plutella xylostella granulovirus 98383 Woodruffvirus TP1604 1982746 Dyoxipapillo mavirus 1 1513255 Poa semilatent virus 12328 Woodruffvirus YDN12 1982747 Dyoxipapillo mavirus 2 2169881 Poaceae associated gemycircularvirus 1 1985392 Woolly monkey hepatitis B virus 68416 Dyozetapapill omavirus 1 1177766 Podivirus S05C243 2733948 Woolly monkey sarcoma virus 11970 Eapunavirus Eap1 2733615 Poecivirus A 2560644 Wound tumor virus 10987 East African cassava mosaic Cameroon virus 223262 Pogseptimavirus PG07 2733996 Wphvirus BPS10C 2560329 East African cassava mosaic Kenya virus 393599 Pogseptimavirus VspSw1 2733997 Wphvirus BPS13 1987727 East African cassava mosaic Malawi virus 223264 Poindextervirus BL10 2734196 Wphvirus hakuna 1987729 East African cassava mosaic virus 62079 Poindextervirus rogue 2748760 Wphvirus megatron 1987728 East African cassava mosaic Zanzibar virus 223275 Poinsettia latent virus 305785 Wphvirus WPh 1922328 East Asian Passiflora distortion virus 2734556 Poinsettia mosaic virus 113553 Wuchang cockroach orthophasmavirus 1 1980542 East Asian Passiflora virus 341167 Pokeweed mosaic virus 1220025 Wuhan mivirus 2507319 Eastern chimpanzee simian foamy virus 2170195 Pokrovskaiavirus fHeYen301 2733374 Wuhan mosquito orthophasmavirus 1 1980543 Eastern equine encephalitis virus 11021 Pokrovskaiavirus pv8018 2733375 Wuhan mosquito orthophasmavirus 2 1980544 Eastern kangaroopox virus 2734571 Polar bear mastadenovirus A Wuhanvirus PHB01 2733969 Eastlansingvirus Sf12 2734004 Pollockvirus pollock 2170215 Wuhanvirus PHB02 2733970 Echarate phlebovirus 2734447 Pollyceevirus pollyC 2560679 Wumivirus millepedae 2509286 Echinochloa hoja blanca tenuivirus 42630 Polybotosvirus Atuph07 2560286 Wumpquatrovirus WMP4 400567 Echinochloa ragged stunt virus Polygonum ringspot orthotospovirus 430606 Wumptrevirus WMP3 440250 Eclipta yellow vein alphasatellite 2030126 Pomona bat hepatitis B virus 2049933 Wutai mosquito phasivirus 1980612 Eclipta yellow vein virus 875324 Pongine gammaherpes virus 2 159603 Wyeomyia orthobunyavirus 273350 Eclunavirus EcL1 2560414 Poplar mosaic virus 12166 Xanthophyllomy ces dendrorhous virus L1A 1167690 Ectocarpus fasciculatus virus a 2083183 Popoffvirus pv56 2560283 Xanthophyllomy ces dendrorhous virus L1B 1167691 Ectocarpus siliculosus virus 1 37665 Porcine associated gemycircularvirus 1 1985393 Xapuri mammarenavirus 2734417 Ectocarpus siliculosus virus a Potato virus Y 12216 Xestia c-nigrum granulovirus 51677 Ectromelia virus 12643 Potato yellow blotch virus 2230887 Xiamenvirus RDJL1 1982373 Ectropis obliqua nucleopolyhed rovirus 59376 Potato yellow mosaic Panama virus 223307 Xiamenvirus RDJL2 1982374 Ectropis obliqua virus 1225732 Potato yellow mosaic virus 10827 Xilang striavirus 2560844 Edenvirus eden 2734230 Potato yellow vein virus 103881 Xinzhou mivirus 2507320 Edge Hill virus 64296 Pothos latent virus 44562 Xipapillomavirus 1 10561 Efquatrovirus AL2 2560415 Potosi orthobunyavirus 2560646 Xipapillomavirus 2 1513273 Efquatrovirus AL3 2560416 Poushouvirus Poushou 2560396 Yokohamavirus PEi21 1980942 Efquatrovirus AUEF3 2560417 Pouzolzia golden mosaic virus 1225069 Yokose virus 64294 Efquatrovirus EcZZ2 2560424 Primate T-lymphotropic virus 3 194443 Yoloswagvirus yoloswag 2734158 Efquatrovirus EF3 2560420 Primolicivirus Pf1 2011081 Yongjia uukuvirus 2734607 Efquatrovirus EF4 2560421 Primula malacoides virus 1 1511840 Youcai mosaic virus 228578 Efquatrovirus EfaCPT1 2560425 Priunavirus PR1 2560652 Yunnan orbivirus 306276 Efquatrovirus IME196 2560426 Privet ringspot virus 2169960 Yushanvirus Spp001 2733978 Efquatrovirus LY0322 2560427 Prochlorococc us virus PHM1 Yushanvirus SppYZU05 2733979 Efquatrovirus PMBT2 2560428 Prospect Hill orthohantavirus 1980485 Yuyuevirus beihaiense 2508254 Efquatrovirus SANTOR1 2560429 Protapanteles paleacritae bracovirus Yuyuevirus shaheense 2508255 Efquatrovirus SHEF2 2560430 Providence virus 213633 Zaire ebolavirus 186538 Efquatrovirus SHEF4 2560431 Prune dwarf virus 33760 Zaliv Terpeniya uukuvirus 2734608 Efquatrovirus SHEF5 2560432 Prunus latent virus 2560653 Zantedeschia mild mosaic virus 270478 Eganvirus EtG 2734059 Prunus necrotic ringspot virus 37733 Zarhavirus zahedan 2734410 Eganvirus ev186 29252 Przondovirus KN31 2733672 Zika virus 64320

The cascade assays described herein are particularly well-suited for simultaneous testing of multiple targets. Pools of two to 10,000 target nucleic acids of interest may be employed, e.g., pools of 2-1000, 2-100, 2-50, or 2-10 target nucleic acids of interest. Further testing may be used to identify the specific member of the pool, if warranted.

While the methods described herein do not require the target nucleic acid of interest to be DNA (and in fact it is specifically contemplated that the target nucleic acid of interest may be RNA), it is understood by those in the field that a reverse transcription step to convert target RNA to cDNA may be performed prior to or while contacting the biological sample with the composition.

Nucleic Acid-Guided Nucleases

The cascade assays comprise nucleic acid-guided nucleases in the reaction mix, either provided as a protein, a coding sequence for the protein, or, in many embodiments, in a ribonucleoprotein (RNP) complex. In some embodiments, the one or more nucleic acid-guided nucleases in the reaction mix may be, for example, a Cas nucleic acid-guided nuclease. Any nucleic acid-guided nuclease having both cis- and trans-cleavage activity may be employed, and the same nucleic acid-guided nuclease may be used for both RNP complexes or different nucleic acid-guided nucleases may be used in RNP1 and RNP2. For example, RNP1 and RNP2 may both comprise Cas12a nucleic acid-guided nucleases, or RNP1 may comprise a Cas13 nucleic acid-guided nuclease and RNP2 may comprise a Cas12a nucleic acid-guided nuclease or vice versa. In embodiments where a variant nucleic acid-guided nuclease is employed, only RNP2 will comprise the variant, and RNP1 may comprise either a Cas12a or Cas13 nucleic acid-guided nuclease. In embodiments where a variant nucleic acid-guided nuclease is not employed, either or both RNP1 and RNP2 can comprise a Cas13 nucleic acid-guided nuclease. Note that trans-cleavage activity is not triggered unless and until cis-cleavage activity (i.e., sequence specific activity) is initiated. Nucleic acid-guided nucleases include Type V and Type VI nucleic acid-guided nucleases, as well as nucleic acid-guided nucleases that comprise a RuvC nuclease domain or a RuvC-like nuclease domain but lack an HNH nuclease domain. Nucleic acid-guided nucleases with these properties are reviewed in Makarova and Koonin, Methods Mol. Biol., 1311:47-75 (2015) and Koonin, et al., Current Opinion in Microbiology, 37:67-78 (2020) and updated databases of nucleic acid-guided nucleases and nuclease systems that include newly-discovered systems include BioGRID ORCS (orcs:thebiogrid.org); GenomeCRISPR (genomecrispr.org); Plant Genome Editing Database (plantcrispr.org) and CRISPRCasFinder (crispercas.i2bc.paris-saclay.fr).

The type of nucleic acid-guided nuclease utilized in the method of detection depends on the type of target nucleic acid of interest to be detected. For example, a DNA nucleic acid-guided nuclease (e.g., a Cas12a, Casl4a, or Cas3) should be utilized if the target nucleic acid of interest is a DNA molecule, and an RNA nucleic acid-guided nuclease (e.g., Cas13a or Cas12g) should be utilized if the target nucleic acid of interest is an RNA molecule. Exemplary nucleic acid-guided nucleases include, but are not limited to, Cas RNA-guided DNA nucleic acid-guided nucleases, such as Cas3, Cas12a (e.g., AsCas12a, LbCas12a), Cas12b, Cas12c, Cas12d, Cas12e, Cas14, Cas12h, Cas12i, and Cas12j; Cas RNA-guided RNA nucleic acid-guided nucleases, such as Cas13a (LbaCas13, LbuCas13, LwaCas13), Cas13b (e.g., CccaCasl3b, PsmCasl3b), and Cas12g; and any other nucleic acid (DNA, RNA, or cDNA) targeting nucleic acid-guided nuclease with cis-cleavage activity and collateral trans-cleavage activity. In some embodiments, the nucleic acid-guided nuclease is a Type V CRISPR-Cas nuclease, such as Cas12a, Cas13a, or Cas14a. In some embodiments, the nucleic acid-guided nuclease is a Type I CRISPR-Cas nuclease, such as Cas3. Type II and Type VI nucleic acid-guided nucleases may also be employed.

In an RNP with a single crRNA (i.e., lacking/without a tracrRNA), Cas12a nucleases and related homologs and orthologs interact with a PAM (protospacer adjacent motif) sequence in a target nucleic acid for dsDNA unwinding and R-loop formation. Cas12a nucleases employ a multistep mechanism to ensure accurate recognition of spacer sequences in the target nucleic acid. The WED, REC1 and PAM-interacting (PI) domains of Cas12a nucleases are responsible for PAM recognition and for initiating invasion of the crRNA in the target dsDNA and for R-loop formation. It has been hypothesized that a conserved lysine residue is inserted into the dsDNA duplex, possibly initiating template strand/non-template strand unwinding. (See Jinek, et al, Mol. Cell, 73(3):589-600.e4 (2019).) PAM binding further introduces a kink in the target strand, which further contributes to local strand separation and facilitates base paring of the target strand to the seed segment of the crRNA while the displaced non-target strand is stabilized by interactions with the PAM-interacting domains. (Id.) The variant nucleic acid-guided nucleases disclosed herein and discussed in detail below have been engineered to disrupt one or both of the WED and PI domains to reconfigure the site of unwinding and R-loop formation to, e.g., sterically obstruct dsDNA target nucleic acids from binding to the variant nucleic acid-guided nuclease and/or to minimize strand separation and/or stabilization of the non-target strand. Though contrary to common wisdom, engineering the variant nucleic acid-guided nucleases in this way contributes to a robust and high-fidelity cascade assay.

The variant nucleic acid-guided nucleases disclosed herein are variants of wildtype Type V nucleases LbCas12a (Lachnospriaceae bacterium Cas12a), AsCas 12a (Acidaminococcus sp. BV3L6 Cas12a), CtCas12a (Candidatus Methanoplasma termitum Cas12a), EeCas12a (Eubacterium eligens Cas12a), Mb3Cas12a (Moraxella bovoculi Cas12a), FnCas12a (Francisella novicida Cas12a), FnoCas12a (Francisella tularensis subsp. novicida FTG Cas12a), FbCas12a (Flavobacteriales bacterium Cas12a), Lb4Cas12a (Lachnospira eligens Cas12a), MbCas12a (Moraxella bovoculi Cas12a), Pb2Cas12a (Prevotella bryantii Cas12a), PgCas12a (Candidatus Parcubacteria bacterium Cas12a), AaCas12a (Acidaminococcus sp. Cas12a), BoCas12a (Bacteroidetes bacterium Cas12a), CMaCas12a (Candidatus Methanomethylophilus alvus CMx1201 Cas12a), and to-be-discovered equivalent Cas12a nucleic acid-guided nucleases and homologs and orthologs of these nucleic acid-guided nucleases (and other nucleic acid-guided nucleases that exhibit both cis-cleavage and trans-cleavage activity), where mutations have been made to the PAM interacting domains such that double-stranded DNA (dsDNA) substrates are bound much more slowly to the variant nucleic acid-guided nucleases than to their wildtype nucleic acid-guided nuclease counterpart, yet single-stranded DNA (ssDNA) substrates are bound at the same rate or nearly so as their wildtype nucleic acid-guided nuclease counterpart. The variant nucleic acid-guided nucleases comprise reconfigured domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules to achieve this phenotype and are described in detail below.

Guide RNA (gRNA)

The present disclosure detects a target nucleic acid of interest via a reaction mixture containing at least two guide RNAs (gRNAs) each incorporated into a different RNP complex (i.e., RNP1 and RNP2). Suitable gRNAs include at least one crRNA region to enable specificity in every reaction. The gRNA of RNP1 is specific to a target nucleic acid of interest and the gRNA of RNP2 is specific to an unblocked nucleic acid or a synthesized activating molecule (both described in detail below). As will be clear given the description below, an advantageous feature of the cascade assay is that, with the exception of the gRNA in the RNP1 (i.e., the gRNA specific to the target nucleic acid of interest), the cascade assay components can stay the same (i.e., are identical or substantially identical) no matter what target nucleic acid(s) of interest are being detected, and the gRNA in RNP1 is easily reprogrammable.

Like the nucleic acid-guided nuclease, the gRNA may be provided in the cascade assay reaction mix in a preassembled RNP, as an RNA molecule, or may also be provided as a DNA sequence to be transcribed, in, e.g., a vector backbone. Providing the gRNA in a pre-assembled RNP complex (i.e., RNP1 or RNP2) is preferred if rapid kinetics are preferred. If provided as a gRNA molecule, the gRNA sequence may include multiple endoribonuclease recognition sites (e.g., Csy4) for multiplex processing. Alternatively, if provided as a DNA sequence to be transcribed, an endoribonuclease recognition site may be encoded between neighboring gRNA sequences such that more than one gRNA can be transcribed in a single expression cassette. Direct repeats can also serve as endoribonuclease recognition sites for multiplex processing. Guide RNAs are generally about 20 nucleotides to about 300 nucleotides in length and may contain a spacer sequence containing a plurality of bases and complementary to a protospacer sequence in the target sequence. The gRNA spacer sequence may be 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or more complementary to its intended target nucleic acid of interest.

The gRNA of RNP1 is capable of complexing with the nucleic acid-guided nuclease of RNP1 to perform cis-cleavage of a target nucleic acid of interest (e.g., a DNA or RNA), which triggers non-sequence specific trans-cleavage of other molecules in the reaction mix. Guide RNAs include any polynucleotide sequence having sufficient complementarity with a target nucleic acid of interest (or target sequences generated by unblocking blocked nucleic acid molecules or target sequences generated by synthesizing synthesized activating molecules as described below). Target nucleic acids of interest (describe in detail above) preferably include a protospacer-adjacent motif (PAM), and, following gRNA binding, the nucleic acid-guided nuclease induces a double-stranded break either inside or outside the protospacer region of the target nucleic acid of interest.

In some embodiments, the gRNA (e.g., of RNP1) is an exo-resistant circular molecule that can include several DNA bases between the 5′ end and the 3′ end of a natural guide RNA and is capable of binding a target sequence. The length of the circularized guide for RNP1 can be such that the circular form of guide can be complexed with a nucleic acid-guided nuclease to form a modified RNP1 which can still retain its cis-cleavage i.e., (specific) and trans-cleavage (i.e., non-specific) nuclease activity.

In any of the foregoing embodiments, the gRNA may be a modified or non-naturally occurring nucleic acid molecule. In some embodiments, the gRNAs of the disclosure may further contain a locked nucleic acid (LNA), a bridged nucleic acid (BNA), and/or a peptide nucleic acid (PNA). By way of further example, a modified nucleic acid molecule may contain a modified or non-naturally occurring nucleoside, nucleotide, and/or internucleoside linkage, such as a 2′-O-methyl (2′-O-Me) modified nucleoside, a 2′-fluoro (2′-F) modified nucleoside, and a phosphorothioate (PS) bond, or any other nucleic acid molecule modifications described herein.

Ribonucleoprotein (RNP) Complex

As described above, although the cascade assay “reaction mix” may comprise separate nucleic acid-guided nucleases and gRNAs (or coding sequences therefor), the cascade assays preferably comprise preassembled ribonucleoprotein complexes (RNPs) in the reaction mix, allowing for faster detection kinetics. The present cascade assay employs at least two types of RNP complexes — RNP1 and RNP2 — each type containing a nucleic acid-guided nuclease and a gRNA. RNP1 and RNP2 may comprise the same nucleic acid-guided nuclease or may comprise different nucleic acid-guided nucleases; however, the gRNAs in RNP1 and RNP2 are different and are configured to detect different nucleic acids. In some embodiments, the reaction mixture contains about 1 fM to about 10 µM of a given RNP1, or about 1 pM to about 1 µM of a given RNP1, or about 10 pM to about 500 pM of a given RNP1. In some embodiments the reaction mixture contains about 6 × 10⁴ to about 6 × 10¹² complexes per microliter (µl) of a given RNP1, or about 6 × 10⁶ to about 6 × 10¹⁰ complexes per microliter (µl) of a given RNP1. In some embodiments, the reaction mixture contains about 1 fM to about 500 µM of a given RNP2, or about 1 pM to about 250 µM of a given RNP2, or about 10 pM to about 100 µM of a given RNP2. In some embodiments the reaction mixture contains about 6 × 10⁴ to about 6 × 10¹² complexes per microliter (µl) of a given RNP2 or about 6 × 10⁶ to about 6 × 10¹² complexes per microliter (µl) of a given RNP2. See Example II below describing preassembling RNPs and Examples V and VI below describing various cascade assay conditions where the relative concentrations of RNP2 and the blocked nucleic acid molecules is adjusted as described below.

In any of the embodiments of the disclosure, the reaction mixture includes 1 to about 1,000 different RNPls (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 27, 28, 19, 20, 21, 22, 23, 24, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1,0000 or more RNP1s), where different RNP1s comprise a different gRNA (or crRNA thereof) polynucleotide sequence. For example, a reaction mixture designed for environmental or oncology testing comprises more than one unique RNP1-gRNA (or RNP1-crRNA) ribonucleoprotein complex for the purpose of detecting more than one target nucleic acid of interest. That is, more than one RNP1 may also be present for the purpose of targeting one target nucleic acid of interest from many sources or for targeting more than one target nucleic acid of interest from a single source.

In any of the foregoing embodiments, the gRNA of RNP1 may be homologous or heterologous, relative to the gRNA of other RNP1(s) present in the reaction mixture. A homologous mixture of RNP1 gRNAs has a number of gRNAs with the same nucleotide sequence, whereas a heterologous mixture of RNP1 gRNAs has multiple gRNAs with different nucleotide sequences (e.g., gRNAs targeting different loci, genes, variants, and/or microbial species). Therefore, the disclosed methods of identifying one or more target nucleic acids of interest may include a reaction mixture containing more than two heterologous gRNAs, more than three heterologous gRNAs, more than four heterologous gRNAs, more than five heterologous gRNAs, more than six heterologous gRNAs, more than seven heterologous gRNAs, more than eight heterologous gRNAs, more than nine heterologous gRNAs, more than ten heterologous gRNAs, more than eleven heterologous gRNAs, more than twelve heterologous gRNAs, more than thirteen heterologous gRNAs, more than fourteen heterologous gRNAs, more than fifteen heterologous gRNAs, more than sixteen heterologous gRNAs, more than seventeen heterologous gRNAs, more than eighteen heterologous gRNAs, more than nineteen heterologous gRNAs, more than twenty heterologous gRNAs, more than twenty-one heterologous gRNAs, more than twenty-three heterologous gRNAs, more than twenty-four heterologous gRNAs, or more than twenty-five heterologous gRNAs. Such a heterologous mixture of RNP1 gRNAs in a single reaction enables multiplex testing.

As a first non-limiting example of a heterologous mixture of RNP1 gRNAs, the reaction mixture may contain: a number of RNP1s (RNP1-1s) having a gRNA targeting parainfluenza virus 1; a number of RNPls (RNP1-2s) having a gRNA targeting human metapneumovirus; a number of RNPls (RNP1-3s) having a gRNA targeting human rhinovirus; a number of RNPls (RNP1-4s) having a gRNA targeting human enterovirus; and a number of RNPls (RNP1-5s) having a gRNA targeting coronavirus HKU1. As a second non-limiting example of a heterologous mixture of RNP1 gRNAs, the reaction mixture may contain: a number of RNPls containing a gRNA targeting two or more SARS-Co-V-2 variants, e.g., B.1.1.7, B.1.351, P.1, B.1.617.2, BA.1, BA.2, BA.2.12.1, BA.4, and BA.5 and subvariants thereof.

As another non-limiting example of a heterologous mixture of RNP1 gRNAs, the reaction mixture may contain RNP1s targeting two or more target nucleic acids of interest from organisms that infect grapevines, such as Guignardia bidwellii (RNP1-1), Uncinula necator (RNP1-2), Botrytis cincerea (RNP1-3), Plasmopara viticola (RNP1-4), and Botryotinis fuckleina (RNP1-5).

Reporter Moieties

The cascade assay detects a target nucleic acid of interest via detection of a signal generated in the reaction mix by a reporter moiety. In some embodiments the detection of the target nucleic acid of interest occurs virtually instantaneously. For example, see the results reported in Example VI for assays comprising 3e4 or 30 copies of MRSA target and within 1 minute or less at 3 copies of MRSA target (see, e.g., FIGS. 10B - 10H). Reporter moieties can comprise DNA, RNA, a chimera of DNA and RNA, and can be single stranded, double stranded, or a moiety that is a combination of single stranded portions and double stranded portions.

Depending on the type of reporter moiety used, trans- and/or cis-cleavage by the nucleic acid-guided nuclease in RNP2 releases a signal. In some embodiments, trans-cleavage of stand-alone reporter moieties (e.g., not bound to any blocked nucleic acid molecules or blocked primer molecules) may generate signal changes at rates that are proportional to the cleavage rate, as new RNP2s are activated over time (shown in FIG. 1B and at top of FIG. 4 ). Trans-cleavage by either an activated RNP1 or an activated RNP2 may release a signal. In alternative embodiments and preferably, the reporter moiety may be bound to the blocked nucleic acid molecule, where trans-cleavage of the blocked nucleic acid molecule (or blocked primer molecule) and conversion to an unblocked nucleic acid molecule (or unblocked primer molecule) may generate signal changes at rates that are proportional to the cleavage rate, as new RNP2s are activated over time, thus allowing for real time reporting of results (shown at FIG. 4 , center). In yet another embodiment, the reporter moiety may be bound to a blocked nucleic acid molecule such that cis-cleavage following the binding of the RNP2 to an unblocked nucleic acid molecule releases a PAM distal sequence, which in turn generates a signal at rates that are proportional to the cleavage rate (shown at FIG. 4 , bottom). In this case, activation of RNP2 by cis- (target specific) cleavage of the unblocked nucleic acid molecule directly produces a signal, rather than producing a signal via indiscriminate trans-cleavage activity. Alternatively or in addition, a reporter moiety may be bound to the gRNA.

The reporter moiety may be a synthetic molecule linked or conjugated to a reporter and quencher such as, for example, a TaqMan probe with a dye label (e.g., FAM or FITC) on the 5′ end and a quencher on the 3′ end. The reporter and quencher may be about 20-30 bases apart or less (i.e., 10-11 nm apart or less) for effective quenching via fluorescence resonance energy transfer (FRET). Alternatively, signal generation may occur through different mechanisms. Other detectable moieties, labels, or reporters can also be used to detect a target nucleic acid of interest as described herein. Reporter moieties can be labeled in a variety of ways, including direct or indirect attachment of a detectable moiety such as a fluorescent moiety, hapten, or colorimetric moiety.

Examples of detectable moieties include various radioactive moieties, enzymes, prosthetic groups, fluorescent markers, luminescent markers, bioluminescent markers, metal particles, and protein-protein binding pairs, e.g., protein-antibody binding pairs. Examples of fluorescent moieties include, but are not limited to, yellow fluorescent protein (YFP), green fluorescence protein (GFP), cyan fluorescence protein (CFP), umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, cyanines, dansyl chloride, phycocyanin, and phycoerythrin. Examples of bioluminescent markers include, but are not limited to, luciferase (e.g., bacterial, firefly, click beetle and the like), luciferin, and aequorin. Examples of enzyme systems having visually detectable signals include, but are not limited to, galactosidases, glucorinidases, phosphatases, peroxidases, and cholinesterases. Identifiable markers also include radioactive elements such as ¹²⁵1, ³⁵S, ¹⁴C, or ³H. Reporters can also include a change in pH or charge of the cascade assay reaction mix.

The methods used to detect the generated signal will depend on the reporter moiety or moieties used. For example, a radioactive label can be detected using a scintillation counter, photographic film as in autoradiography, or storage phosphor imaging. Fluorescent labels can be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence can be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like. Enzymatic labels can be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product. Simple colorimetric labels can be detected by observing the color associated with the label. When pairs of fluorophores are used in an assay, fluorophores are chosen that have distinct emission patterns (wavelengths) so that they can be easily distinguished. In some embodiments, the signal can be detected by lateral flow assays (LFAs). Lateral flow tests are simple devices intended to detect the presence or absence of a target nucleic acid of interest in a sample. LFAs can use nucleic acid molecules conjugated nanoparticles (often gold, e.g., RNA-AuNPs or DNA-AuNPs) as a detection probe, which hybridizes to a complementary target sequence. (See FIG. 9 and the description thereof below.) The classic example of an LFA is the home pregnancy test.

Single-stranded, double-stranded or reporter moieties comprising both single- and double-stranded portions can be introduced to show a signal change proportional to the cleavage rate, which increases with every new activated RNP2 complex over time. In some embodiments and as described in detail below, reporter moieties can also be embedded into the blocked nucleic acid molecules (or blocked primer molecules) for real time reporting of results.

For example, the method of detecting a target nucleic acid molecule in a sample using a cascade assay as described herein can involve contacting the reaction mix with a labeled detection ssDNA containing a fluorescent resonance energy transfer (FRET) pair, a quencher/phosphor pair, or both. A FRET pair consists of a donor chromophore and an acceptor chromophore, where the acceptor chromophore may be a quencher molecule. FRET pairs (donor/acceptor) suitable for use include, but are not limited to, EDANS/fluorescein, IAEDANS/fluorescein, fluorescein/tetramethylrhodamine, fluorescein/Cy 5, IEDANS/DABCYL, fluorescein/QSY-7, fluorescein/LC Red 640, fluorescein/Cy 5.5, Texas Red/DABCYL, BODIPY/DABCYL, Lucifer yellow/DABCYL, coumarin/DABCYL, and fluorescein/LC Red 705. In addition, a fluorophore/quantum dot donor/acceptor pair can be used. EDANS is (5-((2-Aminoethyl)amino)naphthalene-1-sulfonic acid); IAEDANS is 5-({2-[(iodoacetyl)amino]ethyl]amino)naphthalene-1-sulfonic acid); DABCYL is 4-(4- dimethylaminophenyl) diazenylbenzoic acid. Useful quenchers include, but are not limited to, BHQ, DABCYL, QSY 7 and QSY 33.

In any of the foregoing embodiments, the reporter moiety may comprise one or more modified nucleic acid molecules, containing a modified nucleoside or nucleotide. In some embodiments the modified nucleoside or nucleotide is chosen from 2′-O-methyl (2′-O-Me) modified nucleoside, a 2′-fluoro (2′-F) modified nucleoside, and a phosphorothioate (PS) bond, or any other nucleic acid molecule modifications described below.

Nucleic Acid Modifications

For any of the nucleic acid molecules described herein (e.g., blocked nucleic acid molecules, blocked primer molecules, gRNAs, template molecules, synthesized activating molecules, and reporter moieties), the nucleic acid molecules may be used in a wholly or partially modified form. Typically, modifications to the blocked nucleic acid molecules, gRNAs, template molecules, reporter moieties, and blocked primer molecules described herein are introduced to optimize the molecule’s biophysical properties (e.g., increasing nucleic acid-guided nuclease resistance and/or increasing thermal stability). Modifications typically are achieved by the incorporation of, for example, one or more alternative nucleosides, alternative sugar moieties, and/or alternative internucleoside linkages.

For example, one or more of the cascade assay components may include one or more of the following nucleoside modifications: 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C═C—CH₃) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and/or 3-deazaguanine and 3-deazaadenine. The nucleic acid molecules described herein (e.g., blocked nucleic acid molecules, blocked primer molecules, gRNAs, reporter molecules, synthesized activating molecules, and template molecules) may also include nucleobases in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and/or 2-pyridone. Further modification of the nucleic acid molecules described herein may include nucleobases disclosed in USPN 3,687,808; Kroschwitz, ed., The Concise Encyclopedia of Polymer Science and Engineering, NY, John Wiley & Sons, 1990, pp. 858-859; Englisch, et al., Angewandte Chemie, 30:613 (1991); and Sanghvi, Chapter 16, Antisense Research and Applications, CRC Press, Gait, ed., 1993, pp. 289-302.

In addition to or as an alternative to nucleoside modifications, the cascade assay components may comprise 2′ sugar modifications, including 2′-O-methyl (2′-O-Me), 2′-methoxyethoxy (2′—O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE), 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, and/or 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylamino-ethoxy-ethyl or 2′-DMAEOE), i.e., 2′—O—CH₂OCH₂N(CH₃)₂. Other possible 2′-modifications that can modify the nucleic acid molecules described herein (i.e., blocked nucleic acid molecules, gRNAs, synthesized activating molecules, reporter molecules, and blocked primer molecules) may include all possible orientations of OH; F; O—, S—, or N-alkyl (mono- or di-); O—, S—, or N-alkenyl (mono- or di-); O—, S— or N-alkynyl (mono- or di-); or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Other potential sugar substituent groups include, e.g., aminopropoxy (—OCH₂CH₂CH₂NH₂), allyl (—CH₂—CH═CH₂), -O-allyl (—O—CH₂—CH═CH₂) and fluoro (F). 2′-sugar substituent groups may be in the arabino (up) position or ribo (down) position. In some embodiments, the 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the interfering RNA molecule, particularly the 3′ position of the sugar on the 3′ terminal nucleoside or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

Finally, modifications to the cascade assay components may comprise internucleoside modifications such as phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.

The Signal Boosting Cascade Assay Employing Blocked Nucleic Acid Molecules

Before getting to the details relating to addressing undesired unwinding of the blocked nucleic acid molecules (or blocked primer molecules), understanding the cascade assay itself is key. FIG. 1B, described above, depicts the cascade assay generally. A specific embodiment of the cascade assay utilizing blocked nucleic acid molecules is depicted in FIG. 2A and described in detail below. In this embodiment, a blocked nucleic acid is used to prevent the activation of RNP2 in the absence of a target nucleic acid of interest. The method in FIG. 2A begins with providing the cascade assay components RNP1 (201), RNP2 (202) and blocked nucleic acid molecules (203). RNP1 (201) comprises a gRNA specific for a target nucleic acid of interest and a nucleic acid-guided nuclease (e.g., Cas 12a or Cas 14 for a DNA target nucleic acid of interest or a Cas 13a for an RNA target nucleic acid of interest) and RNP2 (202) comprises a gRNA specific for an unblocked nucleic acid molecule and a nucleic acid-guided nuclease (again, e.g., Cas 12a or Cas 14 for a DNA unblocked nucleic acid molecule or a Cas 13a for an RNA unblocked nucleic acid molecule). As described above, the nucleic acid-guided nucleases in RNP1 (201) and RNP2 (202) can be the same or different depending on the type of target nucleic acid of interest and unblocked nucleic acid molecule. What is key, however, is that the nucleic acid-guided nucleases in RNP1 and RNP2 may be activated to have trans-cleavage activity following initiation of cis-cleavage activity.

In a first step, a sample comprising a target nucleic acid of interest (204) is added to the cascade assay reaction mix. The target nucleic acid of interest (204) combines with and activates RNP1 (205) but does not interact with or activate RNP2 (202). Once activated, RNP1 binds the target nucleic acid of interest (204) and cuts the target nucleic acid of interest (204) via sequence-specific cis-cleavage, activating non-specific trans-cleavage of other nucleic acids present in the reaction mix, including the blocked nucleic acid molecules (203). At least one of the blocked nucleic acid molecules (203) becomes an unblocked nucleic acid molecule (206) when the blocking moiety (207) is removed. As described below, “blocking moiety” may refer to nucleoside modifications, topographical configurations such as secondary structures, and/or structural modifications.

Once at least one of the blocked nucleic acid molecules (203) is unblocked, the unblocked nucleic acid molecule (206) can then bind to and activate an RNP2 (208). Because the nucleic acid-guided nucleases in the RNPls (205) and RNP2s (208) have both cis- and trans-cleavage activity, the trans-cleavage activity causes more blocked nucleic acid molecules (203) become unblocked nucleic acid molecules (206) triggering activation of even more RNP2s (208) and more trans-cleavage activity in a cascade. FIG. 2A at bottom depicts the concurrent activation of reporter moieties. Intact reporter moieties (209) comprise a quencher (210) and a fluorophore (211) linked by a nucleic acid sequence. As described above in relation to FIG. 1B, the reporter moieties are also subject to trans-cleavage by activated RNP1 (205) and RNP2 (208). The intact reporter moieties (209) become activated reporter moieties (212) when the quencher (210) is separated from the fluorophore (211), emitting a fluorescent signal (213). Signal strength increases rapidly as more blocked nucleic acid molecules (203) become unblocked nucleic acid molecules (206) triggering cis-cleavage activity of more RNP2s (208) and thus more trans-cleavage activity of the reporter moieties (209). Again, the reporter moieties are shown here as separate molecules from the blocked nucleic acid molecules, but other configurations may be employed and are discussed in relation to FIG. 4 . One particularly advantageous feature of the cascade assay is that, with the exception of the gRNA in the RNP1 (gRNA1), the cascade assay components are modular in the sense that the components stay the same no matter what target nucleic acid(s) of interest are being detected.

FIG. 2B is a diagram showing an exemplary blocked nucleic acid molecule (220) and an exemplary technique for unblocking the blocked nucleic acid molecules described herein. A blocked single-stranded or double-stranded, circular or linear, DNA or RNA molecule (220) comprising a target strand (222) may contain a partial hybridization with a complementary non-target strand nucleic acid molecule (224) containing unhybridized and cleavable secondary loop structures (226) (e.g., hairpin loops, tetraloops, pseudoknots, junctions, kissing hairpins, internal loops, bulges, and multibranch loops). Trans-cleavage of the loops by, e.g., activated RNPls or RNP2s, generates short strand nucleotide sequences or regions (228) which, because of the short length and low melting temperature T_(m) can dehybridize at room temperature (e.g., 15°-25° C.), thereby unblocking the blocked nucleic acid molecule (220) to create an unblocked nucleic acid molecule (230), enabling the internalization of the unblocked nucleic acid molecule (230) (target strand) into an RNP2, leading to RNP2 activation.

A blocked nucleic acid molecule may be single-stranded or double-stranded, circular or linear, and may further contain a partially hybridized nucleic acid sequence containing cleavable secondary loop structures, as exemplified by “L” in FIGS. 2C - 2E. Such blocked nucleic acid molecules typically have a low binding affinity, or high dissociation constant (K_(d)) in relation to binding to RNP2 and may be referred to herein as a high K_(d) nucleic acid molecule. In the context of the present disclosure, the binding of blocked or unblocked nucleic acid molecules or blocked or unblocked primer molecules to RNP2, low K_(d) values range from about 100 fM to about 1 aM or lower (e.g., 100 zM) and high K_(d) values are in the range of 100 nM to about 10-100 10 mM and thus are about 10⁵-, 10⁶-, 10⁷-, 10⁸-, 10⁹- to 10¹⁰-fold or higher as compared to low K_(d) values. Of course, the ideal blocked nucleic acid molecule would have an “infinite K_(d).”

The blocked nucleic acid molecules (high K_(d) molecules) described herein can be converted into unblocked nucleic acid molecules (low K_(d) molecules - also in relation to binding to RNP2) via cleavage of nuclease-cleavable regions (e.g., via active RNPls and RNP2s). The unblocked nucleic acid molecule has a higher binding affinity for the gRNA in RNP2 than does the blocked nucleic acid molecule, although, as described below, there is some “leakiness” where some blocked nucleic acid molecules are able to interact with the gRNA in the RNP2 triggering undesired unwinding.

Once the unblocked nucleic acid molecule is bound to RNP2, the RNP2 activation triggers trans-cleavage activity, which in turn leads to more RNP2 activation by further cleaving blocked nucleic acid molecules, resulting in a positive feedback loop or cascade.

In embodiments where blocked nucleic acid molecules are linear and/or form a secondary structure, the blocked nucleic acid molecules may be single-stranded (ss) or double-stranded (ds) and contain a first nucleotide sequence and a second nucleotide sequence. The first nucleotide sequence has sufficient complementarity to hybridize to a gRNA of RNP2, and the second nucleotide sequence does not. The first and second nucleotide sequences of a blocked nucleic acid molecule may be on the same nucleic acid molecule (e.g., for single-strand embodiments) or on separate nucleic acid molecules (e.g., for double-strand embodiments). Trans-cleavage (e.g., via RNP1 or RNP2) of the second nucleotide sequence converts the blocked nucleic acid molecule to a single-strand unblocked nucleic acid molecule. The unblocked nucleic acid molecule contains only the first nucleotide sequence, which has sufficient complementarity to hybridize to the gRNA of RNP2, thereby activating the trans-cleavage activity of RNP2.

In some embodiments, the second nucleotide sequence at least partially hybridizes to the first nucleotide sequence, resulting in a secondary structure containing at least one loop (e.g., hairpin loops, tetraloops, pseudoknots, junctions, kissing hairpins, internal loops, bulges, and multibranch loops). Such loops block the nucleic acid molecule from binding or incorporating into an RNP complex thereby initiating cis- or trans-cleavage (see, e.g., the exemplary structures in FIGS. 2C - 2F).

In some embodiments, the blocked nucleic acid molecule may contain a protospacer adjacent motif (PAM) sequence, or partial PAM sequence, positioned between the first and second nucleotide sequences, where the first sequence is 5′ to the PAM sequence, or partial PAM sequence, (see FIG. 2G). Inclusion of a PAM sequence may increase the reaction kinetics internalizing the unblocked nucleic acid molecule into RNP2 and thus decrease the time to detection. In other embodiments, the blocked nucleic acid molecule does not contain a PAM sequence.

In some embodiments, the blocked nucleic acid molecules (i.e., high K_(d) nucleic acid molecules in relation to binding to RNP2) of the disclosure may include a structure represented by Formula I (e.g., FIG. 2C), Formula II (e.g., FIG. 2D), Formula III (e.g., FIG. 2E), or Formula IV (e.g., FIG. 2F) wherein Formulas I-IV are in the 5′-to-3′ direction:

-   wherein A is 0-15 nucleotides in length; -   B is 4-12 nucleotides in length; -   L is 3-25 nucleotides in length; -   J is an integer between 1 and 10; -   C is 4-15 nucleotides in length; -   M is 1-25 nucleotides in length or is absent, wherein if M is absent     then A-(B-L)J-C and T-D are separate nucleic acid strands; -   T is 17-135 nucleotides in length (e.g., 17-100, 17-50, or 17-25)     and comprises a sequence complementary to B and C; and -   D is 0-10 nucleotides in length and comprises a sequence     complementary to A;

-   wherein D is 0-10 nucleotides in length; -   T-T′ is 17-135 nucleotides in length (e.g., 17-100, 17-50, or     17-25); -   T′ is 1-10 nucleotides in length and does not hybridize with T; -   C is 4-15 nucleotides in length and comprises a sequence     complementary to T; -   L is 3-25 nucleotides in length and does not hybridize with T; -   B is 4-12 nucleotides in length and comprises a sequence     complementary to T; -   J is an integer between 1 and 10; -   A is 0-15 nucleotides in length and comprises a sequence     complementary to D;

-   wherein T is 17-135 nucleotides in length (e.g., 17-100, 17-50, or     17-25); -   D is 0-10 nucleotides in length; -   M is 1-25 nucleotides in length or is absent, wherein if M is absent     then T-D and A-(B-L)J-C are separate nucleic acid strands; -   A is 0-15 nucleotides in length and comprises a sequence     complementary to D; -   B is 4-12 nucleotides in length and comprises a sequence     complementary to T; -   L is 3-25 nucleotides in length; -   J is an integer between 1 and 10; and -   C is 4-15 nucleotides in length;

-   wherein T is 17-31 nucleotides in length (e.g., 17-100, 17-50, or     17-25); -   D is 0-15 nucleotides in length; -   M is 1-25 nucleotides in length; -   A is 0-15 nucleotides in length and comprises a sequence     complementary to D; and -   L is 3-25 nucleotides in length; -   p is 0 or 1; -   C is 4-15 nucleotides in length and comprises a sequence     complementary to T.

In alternative embodiments of any of these molecules, T (or T-T′) can have a maximum length of 1000 nucleotides, e.g., at most 750, at most 500, at most 400, at more 300, at most 250, at most 200, at most 150, at most 135, at most 100, at most 75, at most 50, or at most 25 nucleotides.

Nucleotide mismatches can be introduced in any of the above structures containing double-strand segments (for example, where M is absent in Formula I or Formula III) to reduce the melting temperature (T_(m)) of the segment such that once the loop (L) is cleaved, the double-strand segment is unstable and dehybridizes rapidly. The percentage of nucleotide mismatches of a given segment may vary between 0% and 50%; however, the maximum number of nucleotide mismatches is limited to a number where the secondary loop structure still forms. “Segments” in the above statement refers to A, B, and C. In other words, the number of hybridized bases can be less than or equal to the length of each double-strand segment and vary based on number of mismatches introduced.

In any blocked nucleic acid molecule having the structure of Formula I, III, or IV, T will have sequence complementarity to a nucleotide sequence (e.g., a spacer sequence) within a gRNA of RNP2. The nucleotide sequence of T is to be designed such that hybridization of T to the gRNA of RNP2 activates the trans-nuclease activity of RNP2. In any blocked nucleic acid molecule having structure of Formula II, T-T′ will have sequence complementarity to a sequence (e.g., a spacer sequence) within the gRNA of RNP2. The nucleotide sequence of T-T′ is to be designed such that hybridization of T-T′ to the gRNA of RNP2 activates the trans-nuclease activity of RNP2. For T or T-T′, full complementarity to the gRNA is not necessarily required, provided there is sufficient complementarity to cause hybridization and trans-cleavage activation of RNP2.

In any of the foregoing embodiments, the blocked nucleic acid molecules of the disclosure may and preferably do further contain a reporter moiety attached thereto such that cleavage of the blocked nucleic acid releases a signal from the reporter moiety. (See FIG. 4 , mechanisms depicted at center and bottom.)

Also, in any of the foregoing embodiments, the blocked nucleic acid molecule may be a modified or non-naturally occurring nucleic acid molecule. In some embodiments, the blocked nucleic acid molecules of the disclosure may further contain a locked nucleic acid (LNA), a bridged nucleic acid (BNA), and/or a peptide nucleic acid (PNA). The blocked nucleic acid molecule may contain a modified or non-naturally occurring nucleoside, nucleotide, and/or internucleoside linkage, such as a 2′-O-methyl (2′-O-Me) modified nucleoside, a 2′-fluoro (2′-F) modified nucleoside, and a phosphorothioate (PS) bond, any other nucleic acid molecule modifications described above, and any combination thereof.

FIG. 2G at left shows an exemplary single-strand blocked nucleic acid molecule and how the configuration of this blocked nucleic acid molecule is able to prevent (or significantly prevent) undesired unwinding of the blocked nucleic acid molecule (or blocked primer molecule) and R-loop formation with an RNP complex, thereby blocking activation of the trans-cleavage activity of RNP2. The single-strand blocked nucleic acid molecule is self-hybridized and comprises: a target strand (TS) sequence complementary to the gRNA (e.g., crRNA) of RNP2; a cleavable non-target strand (NTS) sequence that is partially hybridized (e.g., it contains secondary loop structures) to the TS sequence; and a protospacer adjacent motif (PAM) sequence (e.g., 5′ NAAA 3′) that is specifically located at the 3′ end of the TS sequence. An RNP complex with 3′ → 5′ diffusion (e.g., 1D diffusion) initiates R-loop formation upon PAM recognition. R-loop formation is completed upon a stabilizing ≥17 base hybridization of the TS to the gRNA of RNP2; however, because of the orientation of the PAM sequence relative to the secondary loop structure(s), the blocked nucleic acid molecule sterically prevents the target strand from hybridizing with the gRNA of RNP2, thereby blocking the stable R-loop formation required for the cascade reaction.

FIG. 2G at right shows the blocked nucleic acid molecule being unblocked via trans-cleavage (e.g., by RNP1) and subsequent dehybridization of the non-target strand’s secondary loop structures, followed by binding of the target strand to the gRNA of RNP2, thereby completing stable R-loop formation and activating the trans-cleavage activity of the RNP2 complex.

In some embodiments, the blocked nucleic acid molecules provided herein are circular DNAs, RNAs or chimeric (DNA-RNA) molecules (FIG. 2H), and the blocked nucleic acid molecules may include different base compositions depending on the Cas enzyme used for RNP1 and RNP2. For the circular design of blocked nucleic acid molecules, the 5′ and 3′ ends are covalently linked together. This configuration makes internalization of the blocked nucleic acid molecule into RNP2 — and subsequent RNP2 activation — sterically unfavorable, thereby blocking the progression of the cascade assay. Thus, RNP2 activation (e.g., trans-cleavage activity) happens after cleavage of a portion of the blocked nucleic acid molecule followed by linearization and internalization of unblocked nucleic acid molecule into RNP2.

In some embodiments, the blocked nucleic acid molecules are topologically circular molecules with 5′ and 3′ portions hybridized to each other using DNA, RNA, LNA, BNA, or PNA bases which have a very high melting temperature (Tm). The high Tm causes the structure to effectively behave as a circular molecule even though the 5′ and 3′ ends are not covalently linked. The 5′ and 3′ ends can also have base non-naturally occurring modifications such as phosphorothioate bonds to provide increased stability.

In embodiments where the blocked nucleic acid molecules are circularized (e.g., circular or topologically circular), as illustrated in FIG. 2H, each blocked nucleic acid molecule includes a first region, which is a target sequence specific to the gRNA of RNP2, and a second region, which is a sequence that can be cleaved by nuclease enzymes of activated RNP1 and/or RNP2. The first region may include a nuclease-resistant nucleic acid sequence such as, for example, a phosphorothioate group or other non-naturally occurring nuclease-resistant base modifications, for protection from trans-nucleic acid-guided nuclease activity. In some embodiments, when the Cas enzyme in both RNP1 and RNP2 is Cas12a, the first region of the blocked nucleic acid molecule includes a nuclease-resistant DNA sequence, and the second region of the blocked nucleic acid molecule includes a cleavable DNA sequence. In other embodiments, when the Cas enzyme in RNP1 is Cas12a and the Cas enzyme in RNP2 is Cas13a, the first region of the blocked nucleic acid molecule includes a nuclease-resistant RNA sequence, and the second region of the blocked nucleic acid molecule includes a cleavable DNA sequence and a cleavable RNA sequence. In yet other embodiments, when the Cas enzyme in RNP1 is Cas13a and the Cas enzyme in RNP2 is Cas12a, the first region of the blocked nucleic acid molecule includes a nuclease-resistant DNA sequence, and the second region of the blocked nucleic acid molecule includes a cleavable DNA sequence and a cleavable RNA sequence. In some other embodiments, when the Cas enzyme in both RNP1 and RNP2 is Cas13a, the first region of the blocked nucleic acid molecule includes a nuclease-resistant RNA sequence, and the second region of the blocked nucleic acid molecule includes a cleavable RNA sequence.

The Signal Boosting Cascade Assay Employing Blocked Primer Molecules

The blocked nucleic acid molecules described above may also be blocked primer molecules. Blocked primer molecules include a sequence complementary to a primer binding domain (PBD) on a template molecule (see description below in reference to FIGS. 3A and 3B) and can have the same general structures as the blocked nucleic acid molecules described above. A PBD serves as a nucleotide sequence for primer hybridization followed by primer polymerization by a polymerase. In any of Formulas I, II, or III described above, the blocked primer nucleic acid molecule may include a sequence complementary to the PBD on the 5′ end of T. The unblocked primer nucleic acid molecule can bind to a template molecule at the PBD and copy the template molecule via polymerization by a polymerase.

Specific embodiments of the cascade assay which utilize blocked primer molecules and are depicted in FIGS. 3A and 3B. In the embodiments using blocked nucleic acid molecules described above, activation of RNP1 by binding of N nucleotides of the target nucleic acid molecules or cis-cleavage of the target nucleic acid molecules initiates trans-cleavage of the blocked nucleic acid molecules which were used to activate RNP2 -that is, the unblocked nucleic acid molecules are a target sequence for the gRNA in RNP2. In contrast, in the embodiments using blocked primers activation of RNP1 and trans-cleavage unblocks a blocked primer molecule that is then used to prime a template molecule for extension by a polymerase, thereby synthesizing synthesized activating molecules that are the target sequence for the gRNA in RNP2.

FIG. 3A is a diagram showing the sequence of steps in an exemplary cascade assay involving circular blocked primer molecules and linear template molecules. At left of FIG. 3A is a cascade assay reaction mix comprising 1) RNPls (301) (only one RNP1 is shown); 2) RNP2s (302); 3) linear template molecules (330) (which is the non-target strand); 4) a circular blocked primer molecule (334) (i.e., a high K_(d) molecule); and 5) a polymerase (338), such as a Φ29 polymerase. The linear template molecule (330) (non-target strand) comprises a PAM sequence (331), a primer binding domain (PBD) (332) and, optionally, a nucleoside modification (333) to protect the linear template molecule (330) from 3′ → 5′ exonuclease activity. Blocked primer molecule (334) comprises a cleavable region (335) and a complement to the PBD (332) on the linear template molecule (330).

Upon addition of a sample comprising a target nucleic acid of interest (304) (capable of complexing with the gRNA in RNP1 (301)), the target nucleic acid of interest (304) is bound by with and activates RNP1 (305) but does not interact with or activate RNP2 (302). Once activated, RNP1 cuts the target nucleic acid of interest (304) via sequence specific cis-cleavage, which activates non-specific trans-cleavage of other nucleic acids present in the reaction mix, including at least one of the blocked primer molecules (334). The circular blocked primer molecule (334) (i.e., a high K_(d) molecule, where high K_(d) relates to binding to RNP2) upon cleavage becomes an unblocked linear primer molecule (344) (a low K_(d) molecule, where low K_(d) relates to binding to RNP2), which has a region (336) complementary to the PBD (332) on the linear template molecule (330) and can bind to the linear template molecule (330).

Once the unblocked linear primer molecule (344) and the linear template molecule (330) are hybridized (i.e., hybridized at the PBD (332) of the linear template molecule (330) and the PBD complement (336) on the unblocked linear primer molecule (344)), 3′ → 5′ exonuclease activity of the polymerase (338) removes the unhybridized single-stranded DNA at the end of the unblocked primer molecule (344) and the polymerase (338) can copy the linear template molecule (330) to produce a synthesized activating molecule (346) which is a complement of the non-target strand, which is the target strand. The synthesized activating molecule (346) is capable of activating RNP2 (302 → 308). As described above, because the nucleic acid-guided nuclease in the RNP2 (308) complex exhibits (that is, possesses) both cis- and trans-cleavage activity, more blocked primer molecules (334) become unblocked primer molecules (344) triggering activation of more RNP2s (308) and more trans-cleavage activity in a cascade. As stated above in relation to blocked and unblocked nucleic acid molecules (both linear and circular), the unblocked primer molecule has a higher binding affinity for the gRNA in RNP2 than does the blocked primer molecule, although there may be some “leakiness” where some blocked primer molecules are able to interact with the gRNA in RNP2. However, an unblocked primer molecule has a substantially higher likelihood than a blocked primer molecule to hybridize with the gRNA of RNP2.

FIG. 3A at bottom depicts the concurrent activation of reporter moieties. Intact reporter moieties (309) comprise a quencher (310) and a fluorophore (311). As described above in relation to FIG. 1B, the reporter moieties are also subject to trans-cleavage by activated RNP1 (305) and RNP2 (308). The intact reporter moieties (309) become activated reporter moieties (312) when the quencher (310) is separated from the fluorophore (311), and the fluorophore emits a fluorescent signal (313). Signal strength increases rapidly as more blocked primer molecules (334) become unblocked primer molecules (344) generating synthesized activating molecules (346) and triggering activation of more RNP2 (308) complexes and more trans-cleavage activity of the reporter moieties (309). Again, here the reporter moieties are shown as separate molecules from the blocked nucleic acid molecules, but other configurations may be employed and are discussed in relation to FIG. 4 . Also, as with the cascade assay embodiment utilizing blocked nucleic acid molecules that are not blocked primers, with the exception of the gRNA in RNP1, the cascade assay components stay the same no matter what target nucleic acid(s) of interest are being detected.

FIG. 3B is a diagram showing the sequence of steps in an exemplary cascade assay involving circular blocked primer molecules and circular template molecules. The cascade assay of FIG. 3B differs from that depicted in FIG. 3A by the configuration of the template molecule. Where the template molecule in FIG. 3A was linear, in FIG. 3B the template molecule is circular. At left of FIG. 3B is a cascade assay reaction mix comprising 1) RNPls (301) (only one RNP1 is shown); 2) RNP2s (302); 3) a circular template molecule (352) (non-target strand); 4) a circular blocked primer molecule (334); and 5) a polymerase (338), such as a Φ29 polymerase. The circular template molecule (352) (non-target strand) comprises a PAM sequence (331) and a primer binding domain (PBD) (332). Blocked primer molecule (334) comprises a cleavable region (335) and a complement to the PBD (332) on the circular template molecule (352).

Upon addition of a sample comprising a target nucleic acid of interest (304) (capable of complexing with the gRNA in RNP1 (301)), the target nucleic acid of interest (304) binds to and activates RNP1 (305) but does not interact with or activate RNP2 (302). Once activated, RNP1 cuts the target nucleic acid of interest (304) via sequence specific cis-cleavage, which activates non-specific trans-cleavage of other nucleic acids present in the reaction mix, including at least one of the blocked primer molecules (334). The circular blocked primer molecule (334), upon cleavage, becomes an unblocked linear primer molecule (344), which has a region (336) complementary to the PBD (332) on the circular template molecule (352) and can hybridize with the circular template molecule (352).

Once the unblocked linear primer molecule (344) and the circular template molecule (352) are hybridized (i.e., hybridized at the PBD (332) of the circular template molecule (352) and the PBD complement (336) on the unblocked linear primer molecule (344)), 3′ → 5′ exonuclease activity of the polymerase (338) removes the unhybridized single-stranded DNA at the 3′ end of the unblocked primer molecule (344). The polymerase (338) can now use the circular template molecule (352) (non-target strand) to produce concatenated activating nucleic acid molecules (360) (which are concatenated target strands), which will be cleaved by the trans-cleavage activity of activated RNP1. The cleaved regions of the concatenated synthesized activating molecules (360) (target strand) are capable of activating the RNP2 (302 → 308) complex.

As described above, because the nucleic acid-guided nuclease in RNP2 (308) comprises both cis- and trans-cleavage activity, more blocked primer molecules (334) become unblocked primer molecules (344) triggering activation of more RNP2s (308) and more trans-cleavage activity in a cascade. FIG. 3B at bottom depicts the concurrent activation of reporter moieties. Intact reporter moieties (309) comprise a quencher (310) and a fluorophore (311). As described above in relation to FIG. 1B, the reporter moieties are also subject to trans-cleavage by activated RNP1 (305) and RNP2 (308). The intact reporter moieties (309) become activated reporter moieties (312) when the quencher (310) is separated from the fluorophore (311), and the fluorescent signal (313) is unquenched and can be detected. Signal strength increases rapidly as more blocked primer molecules (334) become unblocked primer molecules (344) generating synthesized activating nucleic acid molecules and triggering activation of more RNP2s (308) and more trans-cleavage activity of the reporter moieties (309). Again, here the reporter moieties are shown as separate molecules from the blocked nucleic acid molecules, but other configurations may be employed and are discussed in relation to FIG. 4 . Also note that as with the other embodiments of the cascade assay, in this embodiment, with the exception of the gRNA in RNP1, the cascade assay components stay the same no matter what target nucleic acid(s) of interest are being detected.

The polymerases used in the “blocked primer molecule” embodiments serve to polymerize a reverse complement strand of the template molecule (non-target strand) to generate a synthesized activating molecule (target strand) as described above. In some embodiments, the polymerase is a DNA polymerase, such as a BST, T4, or Therminator polymerase (New England BioLabs Inc., Ipswich MA., USA). In some embodiments, the polymerase is a Klenow fragment of a DNA polymerase. In some embodiments the polymerase is a DNA polymerase with 5′ → 3′ DNA polymerase activity and 3′ → 5′ exonuclease activity, such as a Type I, Type II, or Type III DNA polymerase. In some embodiments, the DNA polymerase, including the Phi29, T7, Q5®, Q5U®, Phusion®, OneTaq®, LongAmp®, Vent®, or Deep Vent@ DNA polymerases (New England BioLabs Inc., Ipswich MA., USA), or any active portion or variant thereof. Also, a 3′ to 5′ exonuclease can be separately used if the polymerase lacks this activity.

FIG. 4 depicts three mechanisms in which a cascade assay reaction can release a signal from a reporter moiety. FIG. 4 at top shows the mechanism discussed in relation to FIGS. 2A, 3A and 3B. In this embodiment, a reporter moiety 409 is a separate molecule from the blocked nucleic acid molecules present in the reaction mix. Reporter moiety (409) comprises a quencher (410) and a fluorophore (411). An activated reporter moiety (412) emits a signal from the fluorophore (411) once it has been physically separated from the quencher (410).

Reporter Moiety Configurations

FIG. 4 at center shows a blocked nucleic acid molecule (403), which is also a reporter moiety. In addition to quencher (410) and fluorophore (411), a blocking moiety (407) can be seen (see also blocked nucleic acid molecules 203 in FIG. 2A). Blocked nucleic acid molecule/reporter moiety (403) comprises a quencher (410) and a fluorophore (411). In this embodiment of the cascade assay, when the blocked nucleic acid molecule (403) is unblocked due to trans-cleavage initiated by the target nucleic acid of interest binding to RNP1, the unblocked nucleic acid molecule (406) also becomes an activated reporter moiety with fluorophore (411) separated from quencher (410). Note both the blocking moiety (407) and the quencher (410) are removed. In this embodiment, reporter signal is directly generated as the blocked nucleic acid molecules become unblocked. Embodiments of this schema can be used to supply the bulky modifications to the blocked nucleic acid molecules described below.

FIG. 4 at the bottom shows that cis-cleavage of an unblocked nucleic acid molecule or a synthesized activating molecule at a PAM distal sequence by RNP2 generates a signal. Shown are activated RNP2 (408), unblocked nucleic acid molecule (461), quencher (410), and fluorophore (411) forming an activated RNP2 with the unblocked nucleic acid/reporter moiety intact (460). Cis-cleavage of the unblocked nucleic acid/reporter moiety (461) results in an activated RNP2 with the reporter moiety activated (462), comprising the activated RNP2 (408), the unblocked nucleic acid molecule with the reporter moiety activated (463), quencher (410) and fluorophore (411). Embodiments of this schema also can be used to supply the bulky modifications to the blocked nucleic acid molecules described below, and in fact a combination of the configurations of reporter moieties shown in FIG. 4 at center and at bottom may be used.

Preventing Undesired Blocked Nucleic Acid Molecule Unwinding

The present disclosure improves upon the signal cascade assay described in USSNs 17/861,207; 17/861,208; and 17/861,209 by addressing the problem with undesired “unwinding” of the blocked nucleic acid molecule. As described above in detail in relation to FIGS. 1B, 2A, 2B, 2G, 3A, 3B, and 4 , the cascade assay is initiated when a target nucleic acid of interest binds to and activates a first pre-assembled ribonucleoprotein complex (RNP1). The gRNA of RNP1 (gRNA1), comprising a sequence complementary to the target nucleic acid of interest, guides RNP1 to the target nucleic acid of interest. Upon binding of the target nucleic acid of interest to RNP1, RNP1 becomes activated, and the target nucleic acid of interest is cleaved in a sequence specific manner (i.e., cis-cleavage) while also triggering non-sequence specific, indiscriminate trans-cleavage activity which unblocks the blocked nucleic acid molecules in the reaction mix. The unblocked nucleic acid molecules can then activate a second pre-assembled ribonucleoprotein complex (RNP2), where RNP2 comprises a second gRNA (gRNA2) comprising a sequence complementary to the unblocked nucleic acid molecules, and at least one of the unblocked nucleic acid molecules is cis-cleaved in a sequence specific manner. Binding of the unblocked nucleic acid molecule to RNP2 leads to cis-cleavage of the unblocked nucleic acid molecule and non-sequence specific, indiscriminate trans-cleavage activity by RNP2, which in turn unblocks more blocked nucleic acid molecules (and reporter moieties) in the reaction mix activating more RNP2s. Each newly activated RNP2 activates more RNP2s, which in turn cleave more blocked nucleic acid molecules and reporter moieties in a reaction cascade, where all or most of the signal generated comes from the trans-cleavage activity of RNP2.

The improvement to the signal boost cascade assay described herein is drawn to preventing undesired unwinding of the blocked nucleic acid molecules in the reaction mix before the blocked nucleic acid molecules are unblocked via trans-cleavage; that is, preventing undesired unwinding that happens not as a result of unblocking due to trans-cleavage subsequent to cis-cleavage of the target nucleic acid of interest or trans-cleavage of unblocked nucleic acid molecules, but due to other factors. For a description of undesired unwinding, please see FIG. 1C and the attendant description herein. Minimizing undesired unwinding serves two purposes. First, preventing undesired unwinding that happens not as a result of designed or engineered unblocking leads to a “leaky” cascade assay system, which in turn leads to non-specific signal generation and false positives.

Second, preventing undesired unwinding limits non-specific interactions between the nucleic acid-guided nucleases (here, the RNP2s) and blocked nucleic acid molecules (i.e., the target nucleic acids for RNP2) such that only blocked nucleic acid molecules that become unblocked due to trans-cleavage activity react with the nucleic acid-guided nucleases. This “fidelity” in the cascade assay leads primarily to desired interactions and limits “wasteful” interactions where the nucleic acid-guided nucleases are essentially interacting with blocked nucleic acid molecules rather than interacting with unblocked nucleic acid molecules. That is, if unwinding is minimized the nucleic acid-guided nucleases are focused on desired interactions which then leads to immediate signal generation in the cascade assay. Preventing undesired unwinding leads to a more efficient cascade assay system providing more accurate quantification yet with the rapid results characteristic of the cascade assay (see FIGS. 10A - 10H and 12 below).

Ratio of RNP2 to Blocked Nucleic Acid Molecules or Blocked Primers

In one modality to prevent undesired unwinding, the present disclosure describes using an unconventional ratio of blocked nucleic acid molecule (i.e., the target molecule for RNP2) and an RNP complex, here RNP2. The unconventional ratio may be used along with the blocked nucleic acid molecules and RNP2s described above as a primary method for minimizing unwinding or may be used in combination with the other modalities described below to minimize unwinding even more. For example, if one were to design an ideal blocked nucleic acid molecule having an “infinite K_(d)” such as, e.g., through design of the blocked nucleic acid molecule (or blocked primer molecule) and/or inclusion of bulky modifications on the blocked nucleic acid molecule (or blocked primer molecule), the ratio of blocked nucleic acid molecules to RNP2s would not affect the reaction mix to any discernable degree. The common wisdom of the ratio of enzyme to target (here, RNP2 to blocked nucleic acid molecule) is that results are achieved — a signal is generated — when there is a high concentration of nucleic acid-guided nuclease (i.e., RNP complex) and a lower concentration of target or, stated another way, when there is a significant excess of nucleic acid-guided nuclease to target. As described above, in CRISPR detection/diagnostic assay protocols known to date, the CRISPR enzyme (i.e., nucleic acid-guided nuclease) is far in excess of blocked nucleic acid molecules (see, Sun, et al., J. of Translational Medicine, 12:74 (2021); Broughton, et al., Nat. Biotech., 38:870-74 (2020); and Lee, et al., PNAS, 117(41):25722-31 (2020)). However, in a cascade assay system where the nucleic acid-guided nuclease (or RNP complex) is in excess of the targets (here, the blocked nucleic acid molecules), the nucleic acid-guided nucleases encounter the blocked nucleic acid molecules repeatedly, probing the blocked nucleic acid molecules and subjecting them to unwinding. If the blocked nucleic acid molecules are probed and unwound repeatedly, they finally unwind which then triggers activation of RNP2 and cis-cleavage of the blocked nucleic acid molecule even in the absence of a target nucleic acid of interest and the trans-cleavage activity generated thereby.

However, by adjusting the ratio of RNP2 to blocked nucleic acid molecules such that there is an excess of blocked nucleic acid molecules to RNP2, any one blocked nucleic acid molecule may be probed by RNP2; however, the likelihood that any one blocked nucleic acid molecule will be probed repeatedly (and thus unwound) is much lower. If a blocked nucleic acid molecule is probed but then has time to re-hybridize or “recover”, that blocked nucleic acid molecule will stay blocked, will not be subject to non-specific unwinding, and will not trigger activation of RNP2. That is, how often any one blocked nucleic acid molecule is probed is important. As long as an improperly probed blocked nucleic acid has time to re-hybridize after unwinding, there is far less chance that the blocked nucleic acid will be unblocked (i.e., unwound) and will trigger signal generation. That is, preventing non-specific unwinding of the blocked nucleic acid molecules makes the nucleic acid-guided nuclease available for desired unwinding interactions.

In order to prevent non-specific unwinding as described herein, the ratio of blocked nucleic acid molecules to RNP2 should be about 50:1, or about 40:1, or about 35:1, or about 30:1, or about 25:1, or about 20:1, or about 15:1, or about 10:1, or about 7.5:1, or about 5:1, or about 4:1, or about 3:1, or about 2.5:1, or about 2:1, or about 1.5:1, or at least where the molar concentration of blocked nucleic acid molecules is equal to or greater than the molar concentration of RNP2s. As noted above, the signal amplification cascade assay reaction mixture typically contains about 1 fM to about 1 mM of a given RNP2, or about 1 pM to about 500 µM of a given RNP2, or about 10 pM to about 100 µM of a given RNP2; thus, the signal amplification cascade assay reaction mixture typically contains about 2.5 fM to about 2.5 mM blocked nucleic acid molecules, or about 2.5 pM to about 1.25 mM blocked nucleic acid molecules, or about 25 pM to about 250 µM blocked nucleic acid molecules. That is, the reaction mixture contains about 6 × 10⁴ to about 6 × 10¹⁴ RNP2s per microliter (µl) or about 6 × 10⁶ to about 6 × 10¹² RNP2s per microliter (µl) and thus about 6 × 10⁴ to about 6 × 10¹⁴ RNP2s per microliter (µl) or about 6 × 10⁶ to about 6 × 10¹² blocked nucleic acid molecules per microliter (µl). Note, the ratios may be used along with the blocked nucleic acid molecules and RNP2s described above as a primary method for minimizing unwinding or the ratios of blocked nucleic acid molecules to RNP2s may be used in combination with the other modalities described below to further minimize unwinding. Again, if one were to design an ideal blocked nucleic acid molecule having an “infinite K_(d)”, the ratio of blocked nucleic acid molecules to RNP2s would not affect the reaction mix to any discernable degree and the ratios of blocked nucleic acid molecules to RNP2s would not necessarily be within these ranges.

Variant Engineered Nucleic Acid-Guided Nucleases

In some embodiments, the protein sequence of the Cas12a nucleic acid-guided nuclease is modified, with e.g., mutations to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules (see Shin et al., Front. Genet., 11:1577 (2021); doi: 10.3389/fgene.2020.571591, herein incorporated by reference; and Yamano et al., Mol. Cell, 67(4): 633-645 (2017); doi: 10.1016/j.molcel.2017.06.035, herein incorporated by reference) such that the variant engineered nucleic acid-guided nuclease has reduced (or absent) PAM specificity, relative to the unmodified or wildtype nucleic acid-guided nuclease and reduced cleavage activity in relation to double strand DNA with or without a PAM. Such enzymes are referred to herein as single-strand-specific Cas12a nucleic acid-guided nucleases or variant engineered nucleic acid-guided nucleases.

FIG. 5 is a simplified block diagram of an exemplary method 500 for designing, synthesizing and screening variant nucleic acid-guided nucleases. In a first step, mutations or modifications to a nucleic acid-guided nuclease are designed 502, based on, e.g., homology to related nucleic acid-guided nucleases, predicted protein structure and active site configuration, and mutagenesis modeling. For assessment of homologies to other nucleic acid-guided nucleases, amino acid sequences may be found in publicly available databases known to those with skill in the art, including, e.g., Protein DataBank Europe (PDBe), Protein Databank Japan (PDBj), SWISS-PROT, GenBank, RefSeq, TrEMBL, PROSITE, DisProt, InterPro, PIR-International, and PRF/SEQDB. Amino acid homology alignments for purposes of determining similarities to known nucleic acid-guided nucleases can be performed using CUSTALW, CUSTAL OMEGA, COBALT: Multiple Alignment Tool; SIM; and PROBCONS.

For protein engineering and amino acid substitution model predictions for each of the desired mutations, protein modeling software such as SWISS-MODEL, HHpred, I-TASSER, IntFOLD, RaptorX, FoldX, Rosetta, and trRosetta may be used to simulate the structural change(s) and to calculate various parameters due to the structural changes as a result of the amino acid substitution(s), including root mean square deviation (RMSD) value in Angstrom units (i.e., a measurement of the difference between the backbones of the initial nucleic acid-guided nuclease and the mutated nucleic acid nucleic acid-guided nuclease) and changes to the number of hydrogen bonds and conformation in the active site. For the methods used to generate the variant engineered nucleic acid-guided nucleases described herein, see Example VII below.

Following modelling, coding sequences for the variant nucleic acid-guided nucleases that appear to deliver desired properties are synthesized and inserted into an expression vector 504. Methods for site-directed mutagenesis are known in the art, including PCR-based methods such as traditional PCR, where primers are designed to include the desired change; primer extension, involving incorporating mutagenic primers in independent nested PCR before combining them in the final product; and inverse PCR. Additionally, CRISPR gene editing may be performed to introduce the desired mutation or modification to the nucleic acid-guided nuclease coding sequence. The mutated (variant) coding sequences are inserted into an expression vector backbone comprising regulatory sequences such as enhancer and promoter regions. The type of expression vector (e.g., plasmid or viral vector) will vary depending on the type of cells to be transformed.

At step 506, cells of choice are transformed with the variant expression vectors. A variety of delivery systems may be used to introduce (e.g., transform or transfect) the expression vectors into a host cell, including the use of yeast systems, lipofection systems, microinjection systems, biolistic systems, virosomes, liposomes, immunoliposomes, polycations, lipid:nucleic acid conjugates, virions, artificial virions, viral vectors, electroporation, cell permeable peptides, nanoparticles, nanowires, exosomes. Once cells are transformed (or transfected), the transformants are allowed to recover and grow.

Following transformation, the cells are screened for expression of nucleic acid-guided nucleases with desired properties 508, such as cut activity or lack thereof, paste activity or lack thereof, PAM recognition or changes thereto, stability and the ability to form RNPs at various temperatures, and/or cis- and trans-cleavage activity at various temperatures. The assays used to screen the variant nucleic acid-guided nucleases will vary depending on the desired properties, but may include in vitro and in vivo PAM depletion, assays for editing efficiency such as a GFP to BFP assay, and, as used to assess the variant nucleic acid-guided nucleases described herein, in vitro transcription/translation (IVTT) assays were used to measure in vitro trans cleavage with both dsDNA and ssDNA and with and without the presence of a PAM in the blocked nucleic acid molecules, where dsDNA should not activate trans-cleavage regardless of the presence of PAM sequence.

After screening the variant nucleic acid-guided nucleases via the IVTT assays, variants with the preferred properties are identified and selected 510. At this point, a variant may be chosen 512 to go forward into production for use in, e.g., the CRISPR cascade systems described herein; alternatively, promising mutations and/or modifications may be combined 514 and the construction, screening and identifying process is repeated.

In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease may not recognize one or more of the following PAM or partial PAM sequences (listed from 5′ to 3′): TTTN, TTTV, CTTA, CTTV, TCTV, TTCV, YTV, or YTN wherein “A” represents adenine, “C” represents cytosine, “T” represents thymine, “G” represents guanine, “V” represents guanine or cytosine or adenine, “Y” represents guanine or adenine, and “N” represents any nucleotide. In some embodiments, the Cas12a nucleic acid-guided nuclease may have reduced recognition for one or more of the following PAM or partial PAM sequences (listed from 5′ to 3′): TTTN, TTTV, CTTA, CTTV, TCTV, TTCV, YTV, or YTN. The single-strand-specific Cas12a nucleic acid-guided nucleases described herein may have at least 50% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, such as about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%) reduced recognition (i.e., specificity) for one or more of the following PAM or partial PAM sequences (listed from 5′ to 3′): TTTN, TTTV, CTTA, CTTV, TCTV, TTCV, YTV, or YTN.

Exemplary wild type (WT) Cas 12a protein sequences are described in Table 7 below. FIG. 6A shows the result of protein structure prediction using Rosetta and SWISS modeling of wildtype LbCas12a (Lachnospriaceae bacterium Cas12a), and FIG. 6B shows the result of example mutations on the LbCas12a protein structure prediction using Rosetta and SWISS modeling of LbCas12a and indicating the PAM regions (described in more detail in relation to Example VII). Any of these sequences (e.g., SEQ ID NOs: 1-15 and homologs or orthologs thereof) may be modified, as described herein, to generate a single-strand-specific nucleic acid-guided nuclease.

TABLE 7 Exemplary wild type Cas12a nucleic acid-guided nucleases Species Name Reference ID SEQ ID NO: Protein Sequence Lachnospiraceae bacterium Cas12a (LbCas12a) PDD: 6KL9_A SEQ ID NO: 1 MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAED YKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENK ELENLEINLRKEIAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIAL VNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISNM DIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGI DVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQV LSDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKN FDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLKK KAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQ KVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKDLLDSVKS FENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYV TQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYL AIMDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSK KWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKLIDFFKDSISR YPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEV DKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHG QIRLSGGAELFMRRASLKKEELVVHPANSPIANKNPDNPKKTTTLS YDVYKDKRFSEDQYELHIPIAINKCPKNIFKINTEVRVLLKHDDNPY VIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDYHSL LDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAV IALEDLNSGFKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPC ATGGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVN LLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTDADY IKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGI NYQQGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDF LISPVKNSDGIFYDSRNYEAQENAILPKNADANGAYNIARKVLWAI GQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKH Acidaminococcus sp. Cas12a (AsCas12a) NCBI Ref.: WP_021736722.1 SEQ ID NO: 2 MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDH YKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETR NALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFN GKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDI STAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFV STSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVL NLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVI QSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSAL CDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIIS AAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQL DSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKA RNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNG LYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIP KCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKK FQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRP SSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIY NKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRP KSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLS HDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAA NSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRS LNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVI HEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLID KLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVP APYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGD FILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGK RIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLEN DDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDS RFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISN QDWLAYIQELRN Candidatus Methanoplasma termitum (CtCas12a) NCBI Gene ID: 24818655 SEQ ID NO: 3 MNNYDEFTKLYPIQKTIRFELKPQGRTMEHLETFNFFEEDRDRAEK YKILKEAIDEYHKKFIDEHLTNMSLDWNSLKQISEKYYKSREEKDK KVFLSEQKRMRQEIVSEFKKDDRFKDLFSKKLFSELLKEEIYKKGN HQEIDALKSFDKFSGYFIGLHENRKNMYSDGDEITAISNRIVNENFP KFLDNLQKYQEARKKYPEWIIKAESALVAHNIKMDEVFSLEYFNK VLNQEGIQRYNLALGGYVTKSGEKMMGLNDALNLAHQSEKSSKG RIHMTPLFKQILSEKESFSYIPDVFTEDSQLLPSIGGFFAQIENDKDG NIFDRALELISSYAEYDTERIYIRQADINRVSNVIFGEWGTLGGLMR EYKADSINDINLERTCKKVDKWLDSKEFALSDVLEAIKRTGNNDA FNEYISKMRTAREKIDAARKEMKFISEKISGDEESIHIIKTLLDSVQQ FLHFFNLFKARQDIPLDGAFYAEFDEVHSKLFAIVPLYNKVRNYLT KNNLNTKKIKLNFKNPTLANGWDQNKVYDYASLIFLRDGNYYLGI INPKRKKNIKFEQGSGNGPFYRKMVYKQIPGPNKNLPRVFLTSTKG KKEYKPSKEIIEGYEADKHIRGDKFDLDFCHKLIDFFKESIEKHKDW SKFNFYFSPTESYGDISEFYLDVEKQGYRMHFENISAETIDEYVEKG DLFLFQIYNKDFVKAATGKKDMHTIYWNAAFSPENLQDVVVKLN GEAELFYRDKSDIKEIVHREGEILVNRTYNGRTPVPDKIHKKLTDY HNGRTKDLGEAKEYLDKVRYFKAHYDITKDRRYLNDKIYFHVPLT LNFKANGKKNLNKMVIEKFLSDEKAHIIGIDRGERNLLYYSIIDRSG KIIDQQSLNVIDGFDYREKLNQREIEMKDARQSWNAIGKIKDLKEG YLSKAVHEITKMAIQYNAIVVMEELNYGFKRGRFKVEKQIYQKFE NMLIDKMNYLVFKDAPDESPGGVLNAYQLTNPLESFAKLGKQTGI LFYVPAAYTSKIDPTTGFVNLFNTSSKTNAQERKEFLQKFESISYSA KDGGIFAFAFDYRKFGTSKTDHKNVWTAYTNGERMRYIKEKKRN ELFDPSKEIKEALTSSGIKYDGGQNILPDILRSNNNGLIYTMYSSFIA AIQMRVYDGKEDYIISPIKNSKGEFFRTDPKRRELPIDADANGAYNI ALRGELTMRAIAEKFDPDSEKMAKLELKHKDWFEFMQTRGD Eubacterium eligens (EeCas12a) NCBI Gene ID: 41356122 SEQ ID NO: 4 MNGNRSIVYREFVGVIPVAKTLRNELRPVGHTQEHIIQNGLIQEDEL RQEKSTELKNIMDDYYREYIDKSLSGVTDLDFTLLFELMNLVQSSP SKDNKKALEKEQSKMREQICTHLQSDSNYKNIFNAKLLKEILPDFI KNYNQYDVKDKAGKLETLALFNGFSTYFTDFFEKRKNVFTKEAVS TSIAYRIVHENSLIFLANMTSYKKISEKALDEIEVIEKNNQDKMGD WELNQIFNPDFYNMVLIQSGIDFYNEICGVVNAHMNLYCQQTKNN YNLFKMRKLHKQILAYTSTSFEVPKMFEDDMSVYNAVNAFIDETE KGNIIGKLKDIVNKYDELDEKRIYISKDFYETLSCFMSGNWNLITGC VENFYDENIHAKGKSKEEKVKKAVKEDKYKSINDVNDLVEKYIDE KERNEFKNSNAKQYIREISNIITDTETAHLEYDDHISLIESEEKADEM KKRLDMYMNMYHWAKAFIVDEVLDRDEMFYSDIDDIYNILENIVP LYNRVRNYVTQKPYNSKKIKLNFQSPTLANGWSQSKEFDNNAIILI RDNKYYLAIFNAKNKPDKKIIQGNSDKKNDNDYKKMVYNLLPGA NKMLPKVFLSKKGIETFKPSDYIISGYNAHKHIKTSENFDISFCRDLI DYFKNSIEKHAEWRKYEFKFSATDSYSDISEFYREVEMQGYRIDW TYISEADINKLDEEGKIYLFQIYNKDFAENSTGKENLHTMYFKNIFS EENLKDIIIKLNGQAELFYRRASVKNPVKHKKDSVLVNKTYKNQL DNGDVVRIPIPDDIYNEIYKMYNGYIKESDLSEAAKEYLDKVEVRT AQKDIVKDYRYTVDKYFIHTPITINYKVTARNNVNDMVVKYIAQN DDIHVIGIDRGERNLIYISVIDSHGNIVKQKSYNILNNYDYKKKLVE KEKTREYARKNWKSIGNIKELKEGYISGVVHEIAMLIVEYNAIIAM EDLNYGFKRGRFKVERQVYQKFESMLINKLNYFASKEKSVDEPGG LLKGYQLTYVPDNIKNLGKQCGVIFYVPAAFTSKIDPSTGFISAFNF KSISTNASRKQFFMQFDEIRYCAEKDMFSFGFDYNNFDTYNITMGK TQWTVYTNGERLQSEFNNARRTGKTKSINLTETIKLLLEDNEINYA DGHDIRIDMEKMDEDKKSEFFAQLLSLYKLTVQMRNSYTEAEEQE NGISYDKIISPVINDEGEFFDSDNYKESDDKECKMPKDADANGAYC IALKGLYEVLKIKSEWTEDGFDRNCLKLPHAEWLDFIQNKRYE Moraxella bovoculi Cas12a (Mb3Cas12a) GenBank: AKG12737.1 SEQ ID NO: 5 MLFQDFTHLYPLSKTVRFELKPIGKTLEHIHAKNFLNQDETMADM YQKVKAILDDYHRDFIADMMGEVKLTKLAEFYDVYLKFRKNPKD DGLQKQLKDLQAVLRKEIVKPIGNGGKYKAGYDRLFGAKLFKDG KELGDLAKFVIAQEGESSPKLAHLAHFEKFSTYFTGFHDNRKNMY SDEDKHTAIAYRLIHENLPRFIDNLQILATIKQKHSALYDQIINELTA SGLDVSLASHLDGYHKLLTQEGITAYNTLLGGISGEAGSRKIQGINE LINSHHNQHCHKSERIAKLRPLHKQILSDGMGVSFLPSKFADDSEV CQAVNEFYRHYADVFAKVQSLFDGFDDYQKDGIYVEYKNLNELS KQAFGDFALLGRVLDGYYVDVVNPEFNERFAKAKTDNAKAKLTK EKDKFIKGVHSLASLEQAIEHYTARHDDESVQAGKLGQYFKHGLA GVDNPIQKIHNNHSTIKGFLERERPAGERALPKIKSDKSPEIRQLKEL LDNALNVAHFAKLLTTKTTLHNQDGNFYGEFGALYDELAKIATLY NKVRDYLSQKPFSTEKYKLNFGNPTLLNGWDLNKEKDNFGVILQK DGCYYLALLDKAHKKVFDNAPNTGKSVYQKMIYKLLPGPNKMLP KVFFAKSNLDYYNPSAELLDKYAQGTHKKGDNFNLKDCHALIDFF KAGINKHPEWQHFGFKFSPTSSYQDLSDFYREVEPQGYQVKFVDIN ADYINELVEQGQLYLFQIYNKDFSPKAHGKPNLHTLYFKALFSEDN LVNPIYKLNGEAEIFYRKASLDMNETTIHRAGEVLENKNPDNPKKR QFVYDIIKDKRYTQDKFMLHVPITMNFGVQGMTIKEFNKKVNQSI QQYDEVNVIGIDRGERHLLYLTVINSKGEILEQRSLNDITTASANGT QMTTPYHKILDKREIERLNARVGWGEIETIKELKSGYLSHVVHQIS QLMLKYNAIVVLEDLNFGFKRGRFKVEKQIYQNFENALIKKLNHL VLKDKADDEIGSYKNALQLTNNFTDLKSIGKQTGFLFYVPAWNTS KIDPETGFVDLLKPRYENIAQSQAFFGKFDKICYNADRGYFEFHIDY AKFNDKAKNSRQIWKICSHGDKRYVYDKTANQNKGATIGVNVND ELKSLFTRYHINDKQPNLVMDICQNNDKEFHKSLMYLLKTLLALR YSNASSDEDFILSPVANDEGVFFNSALADDTQPQNADANGAYHIA LKGLWLLNELKNSDDLNKVKLAIDNQTWLNFAQNR Francisella novicida Cas12a (FnCas12a) UniProtKB/Swiss-Prot: A0Q7Q2.1 SEQ ID NO: 6 MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDY KKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNL QKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLIL WLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRK NVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIK KDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFN TIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQIL SDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSL LFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYIT QQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDI DKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQ ASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFY LVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANG WDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGE GYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTK NGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQR YNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDF SAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIP KKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPIT INFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVDG KGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNI KEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQ VYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKK MGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFD KICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDK NHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFA KLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMP QDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQ NRNN Francisella tularensis subsp. novicida FTG Cas12a (FnoCas12a) NCBI Gene ID: 60806594 SEQ ID NO: 7 MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDY KKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNL QKDFKSAKDTIKKQISKYINDSEKFKNLFNQNLIDAKKGQESDLIL WLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRK NVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIK KDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFN TIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQIL SDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSL LFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYIT QQVAPKNLDNPSKKEQDLIAKKTEKAKYLSLETIKLALEEFNKHRD IDKQCRFEEILSNFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLL QASAEEDVKAIKDLLDQTNNLLHRLKIFHISQSEDKANILDKDEHF YLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLASG WDKNKESANTAILFIKDDKYYLGIMDKKHNKIFSDKAIEENKGEG YKKIVYKQIADASKDIQNLMIIDGKTVCKKGRKDRNGVNRQLLSL KRKHLPENIYRIKETKSYLKNEARFSRKDLYDFIDYYKDRLDYYDF EFELKPSNEYSDFNDFTNHIGSQGYKLTFENISQDYINSLVNEGKLY LFQIYSKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAE LFYRKQSIPKKITHPAKETIANKNKDNPKKESVFEYDLIKDKRFTED KFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLA YYTLVDGKGNIIKQDNFNIIGNDRMKTNYHDKLAAIEKDRDSARK DWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRG RFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTA PFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKS QEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRL INFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICG ESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDS RQAPKNMPQDADANGAYHIGLKGLMLLDRIKNNQEGKKLNLVIK NEEYFEFVQNRNN Flavobacteriales bacterium (FbCas12a) NCBI Gene ID: MBE7442138.1 SEQ ID NO: 8 MKNNNMLNFTNKYQLSKTLRFELKPIGKTKENIIAKNILKKDEERA ESYQLMKKTIDGFHKHFIELAMQEVQKTKLSELEEFAELYNKSAEE KKKDDKFDDKFKKVQEALRKEIVKGFNSEKVKYYYSNIDKKILFT ELLKNWIPNEKMITELSEWNAKTKEEKEHLVYLDKEFENFTTYFG GFHKNRENMYTDKEQSTAIAYRLIHENLPKFLDNINIYKKVKEIPV LREECKVLYKEIEEYLNVNSIDEVFELSYYNKTLTQKDIDVYNLIIG GRTLEEGKKKIQGLNEYINLYNQKQEKKNRIPKLKILYKQILSDRDS ISWLPESFEDDNEKTASQKVLEAINLYYRDNLLCFQPKDKKDTENV LEETKKLLAGLSTSDLSKIYIRNDRAITDISQALFKDYGVIKDALKF QFIQSFTIGKNGLSKKQEEAIEKHLKQKYFSIAEIENALFTYQSETDA LKELKENSHPVVDYFINHFKAKKKEETDKDFDLIANIDAKYSCIKG LLNTPYPKDKKLYQRSKGDNDIDNIKAFLDALMELLHFVKPLALS NDSTLEKDQNFYSHFEPYYEQLELLIPLYNKVRNFAAKKPYSTEKF KLNFDNATLLNGWDKNKETDNTSVILRKDGLYYLAIMPQDNKNV FKDSPDLKANENCFEKMDYKQMALPMGFGAFVRKCFGTASQLG WNCPESCKNEEDKIIIKEDEVKNNRAEIIDCYKDFLNIYEKDGFQYK EYGFDFKESNKYESLREFFIDVEQQGYKITFQNISENYINQLVEDGK LYLFQIYNKDFSPYSKGKPNMHTMYWKALFDSENLKDVVYKLNG QAEVFYRKKSIEQKNIVTHKANEPIDNKNPKAKKKQSTFEYDLIKD KRYTVDKFQFHVPITLNFKATGNDYINQDVLTYLKNNPEVNIIGLD RGERHLIYLTLINQKGEILLQESLNTIVNKKYDIETPYHTLLQNKED ERAKARENWGVIENIKELKEGYISQVVHKIAKLMVEYNAIVVMED LNTGFKRGRFKVEKQVYQKLEKMLIDKLNYLVFKDKDPSEVGGL YHALQLTNKFENFSKIGKQSGFLFYVPAWNTSKIDPTTGFVNLFNT KYESVPKAQEFFKKFKSIKFNSAENYFEFAFDYNDFTTRAEGTKTD WIVCTYGDRIKTFRNPDKVNQWDNQEVNLTEQFEDFFGKNNLIYG DGNCIKNQIILHDKKEFFEGLLHLLKLTLQMRNSITNSEVDYLISPV KNNKGEFYDSRKANNTLPKDADANGAYHIAKKGLVLLNRLKENE VEEFEKSKKVKDGKSQWLPNKDWLDFVQRNVEDMVVV Lachnospira eligens (Lb4Cas12a) NCBI Gene ID: MBS6299380.1 SEQ ID NO: 9 MNGNRSIVYREFVGVTPVAKTLRNELRPVGHTQEHIIQNGLIQEDE LRQEKSTELKNIMDDYYREYIDKSLSGVTDLDFTLLFELMNLVQSS PSKDNKKALEKEQSKMREQICTHLQSDSNYKNIFNAKLFKEILPDFI KNYNQYDVKDKAGKLETVALFNGFSTYFTDFFEKRKNVFTKEAV STSIAYRIVHENSLIFLANMTSYKKISEKALDEIEVIEKNNQDKMGD WELNQIFNPDFYNMVLIQSGIDFYNEICGVVNAHMNLYCQQTRNN YNLFKMRKLHKQILAYTSTSFEVPKMFEDDMSVYNAVNAFIDETE KGNIIVKLKDIVNKYDELDEKRIYISKDFYETLSCFISGNWNLITGC VENFYDENIHAKGKSKEEKVKKAVKEDKYKSINDVNDLVEKYIDE KERNEFKNSNAKQYIREISNIITDTETAHLEYDEHISLIESEEKADEM KKRLDMYMNMYHWAKAFIVDEVLDRDEMFYSDIDDIYNILENIVP LYNRVRNYVTQKPYNSKKIKLNFQSPTLANGWSQSKEFDNNAIILI RDNKYYLAIFNAKNKPDKKIIQGNSDKKNDNDYKKMVYNLLPGA NKMLPKVFLSKKGIETFKPSDYIISGYNAHKHIKTSENFDISFCRDLI DYFKNSIEKHAEWRKYEFKFSATDSYNDISEFYREVEMQGYRIDW TYISEADINKLDEEGKIYLFQIYNKYFAENSTGKENLHTMYFKNIFS EENLKDIIIKLNGQAELFYRRASVKNPVKHKKDSVLVNKTYKNQL DNGDVVRIPIPDDIYNEIYKMYNGYIKESDLSEAAKEYLDKVEVRT AQKDIVKDYRYTVDKYFIHTPITINYKVTARNNVNDMAVKYIAQN DDIHVIGIDRGERNLIYISVIDSHGNIVKQKSYNILNNYDYKKKLVE KEKTREYARKNWKSIGNIKELKEGYISGVVHEIAMLMVEYNAIIA MEDLNYGFKRGRFKVERQVYQKFESMLINKLNYFASKGKSVDEP GGLLRGYQLTYVPDNIKNLGKQCGVIFYVPAAFTSKIDPSTGFISAF NFKSISTNASRKQFFMQFDEIRYCAEKDMFSFGFDYNNFDTYNITM GKTQWTVYTNGERLQSEFNNARRTGKTKSINLTETIKLLLKDNKIN YADGHDVRIDMEKMDEDKNSEFFAQLLSLYKLTVQMRNSYTEAE EQEKGISYDKIISPVINDEGEFFDSDNYKESDDKECKMPKDADANG AYCIALKGLYEVLKIKSEWTEDGFDRNCLKLPHAEWLDFIQNKRY E Moraxella bovoculi (MbCas12a) NCBI Gene ID: WP_046697655.1 SEQ ID NO: 10 MLFQDFTHLYPLSKTVRFELKPIGRTLEHIHAKNFLSQDETMADMY QKVKVILDDYHRDFIADMMGEVKLTKLAEFYDVYLKFRKNPKDD GLQKQLKDLQAVLRKESVKPIGSGGKYKTGYDRLFGAKLFKDGK ELGDLAKFVIAQEGESSPKLAHLAHFEKFSTYFTGFHDNRKNMYS DEDKHTAIAYRLIHENLPRFIDNLQILTTIKQKHSALYDQIINELTAS GLDVSLASHLDGYHKLLTQEGITAYNRIIGEVNGYTNKHNQICHKS ERIAKLRPLHKQILSDGMGVSFLPSKFADDSEMCQAVNEFYRHYT DVFAKVQSLFDGFDDHQKDGIYVEHKNLNELSKQAFGDFALLGR VLDGYYVDVVNPEFNERFAKAKTDNAKAKLTKEKDKFIKGVHSL ASLEQAIEHHTARHDDESVQAGKLGQYFKHGLAGVDNPIQKIHNN HSTIKGFLERERPAGERALPKIKSGKNPEMTQLRQLKELLDNALNV AHFAKLLTTKTTLDNQDGNFYGEFGVLYDELAKIPTLYNKVRDYL SQKPFSTEKYKLNFGNPTLLNGWDLNKEKDNFGVILQKDGCYYLA LLDKAHKKVFDNAPNTGKNVYQKMVYKLLPGPNKMLPKVFFAK SNLDYYNPSAELLDKYAKGTHKKGDNFNLKDCHALIDFFKAGINK HPEWQHFGFKFSPTSSYRDLSDFYREVEPQGYQVKFVDINADYIDE LVEQGKLYLFQIYNKDFSPKAHGKPNLHTLYFKALFSEDNLADPIY KLNGEAQIFYRKASLDMNETTIHRAGEVLENKNPDNPKKRQFVYD IIKDKRYTQDKFMLHVPITMNFGVQGMTIKEFNKKVNQSIQQYDE VNVIGIDRGERHLLYLTVINSKGEILEQRSLNDITTASANGTQVTTP YHKILDKREIERLNARVGWGEIETIKELKSGYLSHVVHQINQLMLK YNAIVVLEDLNFGFKRGRFKVEKQIYQNFENALIKKLNHLVLKDK ADDEIGSYKNALQLTNNFTDLKSIGKQTGFLFYVPAWNTSKIDPET GFVDLLKPRYENIAQSQAFFGKFDKICYNTDKGYFEFHIDYAKFTD KAKNSRQKWAICSHGDKRYVYDKTANQNKGAAKGINVNDELKS LFARYHINDKQPNLVMDICQNNDKEFHKSLMCLLKTLLALRYSNA SSDEDFILSPVANDEGVFFNSALADDTQPQNADANGAYHIALKGL WLLNELKNSDDLNKVKLAIDNQTWLNFAQNR Prevotella bryantii (Pb2Cas12a) NCBI Gene ID: WP_039871282.1 SEQ ID NO: 11 MKFTDFTGLYSLSKTLRFELKPIGKTLENIKKAGLLEQDQHRADSY KKVKKIIDEYHKAFIEKSLSNFELKYQSEDKLDSLEEYLMYYSMKR IEKTEKDKFAKIQDNLRKQIADHLKGDESYKTIFSKDLIRKNLPDFV KSDEERTLIKEFKDFTTYFKGFYENRENMYSAEDKSTAISHRIIHEN LPKFVDNINAFSKIILIPELREKLNQIYQDFEEYLNVESIDEIFHLDYF SMVMTQKQIEVYNAIIGGKSTNDKKIQGLNEYINLYNQKHKDCKL PKLKLLFKQILSDRIAISWLPDNFKDDQEALDSIDTCYKNLLNDGN VLGEGNLKLLLENIDTYNLKGIFIRNDLQLTDISQKMYASWNVIQD AVILDLKKQVSRKKKESAEDYNDRLKKLYTSQESFSIQYLNDCLR AYGKTENIQDYFAKLGAVNNEHEQTINLFAQVRNAYTSVQAILTTP YPENANLAQDKETVALIKNLLDSLKRLQRFIKPLLGKGDESDKDER FYGDFTPLWETLNQITPLYNMVRNYMTRKPYSQEKIKLNFENSTLL GGWDLNKEHDNTAIILRKNGLYYLAIMKKSANKIFDKDKLDNSGD CYEKMVYKLLPGANKMLPKVFFSKSRIDEFKPSENIIENYKKGTHK KGANFNLADCHNLIDFFKSSISKHEDWSKFNFHFSDTSSYEDLSDF YREVEQQGYSISFCDVSVEYINKMVEKGDLYLFQIYNKDFSEFSKG TPNMHTLYWNSLFSKENLNNIIYKLNGQAEIFFRKKSLNYKRPTHP AHQAIKNKNKCNEKKESIFDYDLVKDKRYTVDKFQFHVPITMNFK STGNTNINQQVIDYLRTEDDTHIIGIDRGERHLLYLVVIDSHGKIVE QFTLNEIVNEYGGNIYRTNYHDLLDTREQNREKARESWQTIENIKE LKEGYISQVIHKITDLMQKYHAVVVLEDLNMGFMRGRQKVEKQV YQKFEEMLINKLNYLVNKKADQNSAGGLLHAYQLTSKFESFQKLG KQSGFLFYIPAWNTSKIDPVTGFVNLFDTRYESIDKAKAFFGKFDSI RYNADKDWFEFAFDYNNFTTKAEGTRTNWTICTYGSRIRTFRNQA KNSQWDNEEIDLTKAYKAFFAKHGINIYDNIKEAIAMETEKSFFED LLHLLKLTLQMRNSITGTTTDYLISPVHDSKGNFYDSRICDNSLPAN ADANGAYNIARKGLMLIQQIKDSTSSNRFKFSPITNKDWLIFAQEK PYLND Candidatus Parcubacteria bacterium (PgCas12a) NCBI Gene ID: BCX15829.1 SEQ ID NO: 12 MENKNNQTQSIWSVFTKKYSLQKTLRFELKPVGETKKWLEENDIF KKDLNIDKSYNQAKFYFDKLHQDFIKESLSVENGIRNIDFEKFAKIF ESNKEKIVSLKKKNKEVKDKNKKNWDEISKLEKEIEGQRENLYKEI RELFDKRAEKWKKEYQDKEIERGGKKEKIKFSSADLKQKGVNFLT AAGIINILKYKFPAEKDEEFRKEGYPSLFINDELNPGKKIYIFESFDK FTTYLSKFQQTRENLYKDDGTSTAVATRIVSNFERFLENKSLFEEK YKNKAKDVGLTKEEEKVFEINYYYDCLIQEGIDKYNKIIGEINRKT KEYRDKNKIDKKDLPLFLNLEKQILGEVKKERVFIEAKDEKTEEEV FIDRFQEFIKRNKIKIYGDEKEEIEGAKKFIEDFTSGIFENDYQSIYLK KNVINEIVNKWFSNPEEFLMKLTGVKSEEKIKLKKFTSLDEFKNAIL SLEGDIFKSRFYKNEVNPEAPLEKEEKSNNWENFLKIWRFEFESLFK DKVEKGEIKKDKNGEPIQIFWGYTDKLEKEAEKIKFYSAEKEQIKTI KNYCDAALRINRMMRYFNLSDKDRKDVPSGLSTEFYRLVDEYFN NFEFNKYYNGIRNFITKKPSDENKIKLNFESRSLLDGWDVSKEKDN LGLIFIKNNKYYLGVLRKENSKLFDYQITEKDNQKEKERKNNLKNE ILANDNEDFYLKMNYWQIADPAKDIFNLVLMPDNTVKRFTKLEEK NKHWPDEIKRIKEKGTYKREKVNREDLVKIINYFRKCALIYWKKF DLKLLPSEEYQTFKDFTDHIALQGYKINFDKIKASYIEKQLNDGNL YLFEVSNKDFYKYKKPDSRKNIHTLYWEHIFSKENLEEIKYPLIRLN GKAEIFYRDVLEMNEEMRKPVILERLNGAKQAKREDKPVYHYQR YLKPTYLFHCPITLNADKPSSSFKNFSSKLNHFIKDNLGKINIIGIDR GEKNLLYYCVINQNQEILDYGSLNKINLNKVNNVNYFDKLVEREK QRQLERQSWEPVAKIKDLKQGYISYVVRKICDLIINHNAIVVLEDLS RRFKQIRNGISERTVYQQFEKALIDKLNYLIFKDNRDVFSPGGVLN GYQLAAPFTSFKDIEKAKQTGVLFYTSAEYTSQTDPLTGFRKNIYIS NSASQEKIKELINKLKKFGWDDTEESYFIEYNQVDFAEKKKKPLSK DWTIWTKVPRVIRWKESKSSYWSYKKINLNEEFRDLLEKYGFEAQ SNDILSNLKKRIAENDKLLVEKKEFDGRLKNFYERFIFLFNIVLQVR NTYSLSVEIDKTEKKLKKIDYGIDFFASPVKPFFTTFGLREIGIEKDG KVVKDNAREEIASENLAEFKDRLKEYKPEEKFDADGVGAYNIARK GLIILEKIKNNPNKPDLSISKEEWDKFVQR Acidaminococcus sp. (AaCas12a) NCBI Gene ID: WP_021736722.1 SEQ ID NO: 13 MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDH YKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETR NALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFN GKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDI STAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFV STSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVL NLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVI QSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSAL CDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIIS AAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQL DSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKA RNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNG LYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIP KCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKK FQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRP SSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIY NKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRP KSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLS HDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAA NSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRS LNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVI HEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLID KLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVP APYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGD FILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGK RIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLEN DDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDS RFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISN QDWLAYIQELRN Bacteroidetes bacterium (BoCas12a) NCBI Gene ID: PKP47250.1 SEQ ID NO: 14 MESPTTQLKKFTNLYQLSKTLRFELKPVGKTKEHIETKGILKKDEE RAVNYKLIKKIIDGFHKHFIELAMQQVKLSKLDELAELYNASAERK KEESYKKELEQVQAALRKEIVKGFNIGEAKEIFSKIDKKELFTELLD EWVKNLEEKKLVDDFKTFTTYFTGFHENRKNMYTDKAQSTAIAY RLVHENLPKFLDNTKIFKQIETKFEASKIEEIETKLEPIIQGTSLSEIFT LDYYNHALTQAGIDFINNIIGGYTEDEGKKKIQGLNEYINLYNQKQ EKKNRIPKLKILYKQILSDRDSISFLPDAFEDSQEVLNAIQNYYQTN LIDFKPKDKEETENVLEETKKLLTELFSNELSKIYIRNDKAITDISQA LFNDWGVFKSALEYKFIQDLELGTKELSKKQENEKEKYLKQAYFSI AEIENALFAYQNETDVLNEIKENSHPIADYFTKHFKAKKKVDTSTS SVEKDFDLIANIDAKYSCIKGILNTDYPKDKKLNQEKKTIDDLKVFL DSLMELLHFVKPLALPNDSILEKDENFYSHFESYYEQLELLIPLYNK VRNYAAKKPYSTEKFKLNFENATLLKGWDKNKEIDNTSVILRKRG LYYLAIMPQDNKNVFKKSPNLKNNESCFEKMDYKQMALPMGFGA FVRKCFGTAFQLGWNCPKSCINEEDKIIIKEDEVKNNRAEIIDCYKD FLNIYEKDGFQYKEYGFNFKESKEYESLREFFIDVEQKGYKIEFQNI SENYIHQLVNEGKLYLFQIYNKDFSSYSKGKPNMHTMYWKALFDP ENLKDVVYKLNGQAEVFYRKKSIEDKNIITHKANEPIENKNPKAKK TQSTFEYDLIKDKRYTVDKFHFHVPITINFKATGNNYINQQVLDHL KNNTDVNIIGLDRGERHLIYLTLINQKGEILLQESLNTIVNKKFDIET PYHTLLQNKEDERAKARENWGVIENIKELKEGYLSQVVHKIAKLM VDYNAIVVMEDLNTGFKRGRFKVEKQVYQKLEKMLIDKLNYLVF KDKDPNEVGGLYNALQLTNKFESFSKMGKQSGFLFYVPAWNTSKI DPTTGFVNLFYAKYESIPKAQDFFTKFKSIRYNSDENYFEFAFDYN DFTTRAEGTKSDWTVCTYGDRIKTFRNPEKNNQWDNQEVNLIEQF EAFFGKHNITYGDGNCIKKQLIEQDKKEFFEELFHLFKLTLQMRNSI TNSEIDYLISPVKNSKKEFYDSRKADSTLPKDADANGAYHIAKKGL MWLEKINSFKGSDWKKLDLDKTNKTWLNFVQETASEKHKKLQTV Candidatus Methanomethylop hilus alvus Mx1201 (CMaCas12a) NCBI Gene ID: 15139718 SEQ ID NO: 15 MDAKEFTGQYPLSKTLRFELRPIGRTWDNLEASGYLAEDRHRAEC YPRAKELLDDNHRAFLNRVLPQIDMDWHPIAEAFCKVHKNPGNK ELAQDYNLQLSKRRKEISAYLQDADGYKGLFAKPALDEAMKIAKE NGNESDIEVLEAFNGFSVYFTGYHESRENIYSDEDMVSVAYRITED NFPRFVSNALIFDKLNESHPDIISEVSGNLGVDDIGKYFDVSNYNNF LSQAGIDDYNHIIGGHTTEDGLIQAFNVVLNLRHQKDPGFEKIQFK QLYKQILSVRTSKSYIPKQFDNSKEMVDCICDYVSKIEKSETVERAL KLVRNISSFDLRGIFVNKKNLRILSNKLIGDWDAIETALMHSSSSEN DKKSVYDSAEAFTLDDIFSSVKKFSDASAEDIGNRAEDICRVISETA PFINDLRAVDLDSLNDDGYEAAVSKIRESLEPYMDLFHELEIFSVG DEFPKCAAFYSELEEVSEQLIEIIPLFNKARSFCTRKRYSTDKIKVNL KFPTLADGWDLNKERDNKAAILRKDGKYYLAILDMKKDLSSIRTS DEDESSFEKMEYKLLPSPVKMLPKIFVKSKAAKEKYGLTDRMLEC YDKGMHKSGSAFDLGFCHELIDYYKRCIAEYPGWDVFDFKFRETS DYGSMKEFNEDVAGAGYYMSLRKIPCSEVYRLLDEKSIYLFQIYN KDYSENAHGNKNMHTMYWEGLFSPQNLESPVFKLSGGAELFFRK SSIPNDAKTVHPKGSVLVPRNDVNGRRIPDSIYRELTRYFNRGDCRI SDEAKSYLDKVKTKKADHDIVKDRRFTVDKMMFHVPIAMNFKAI SKPNLNKKVIDGIIDDQDLKIIGIDRGERNLIYVTMVDRKGNILYQD SLNILNGYDYRKALDVREYDNKEARRNWTKVEGIRKMKEGYLSL AVSKLADMIIENNAIIVMEDLNHGFKAGRSKIEKQVYQKFESMLIN KLGYMVLKDKSIDQSGGALHGYQLANHVTTLASVGKQCGVIFYIP AAFTSKIDPTTGFADLFALSNVKNVASMREFFSKMKSVIYDKAEG KFAFTFDYLDYNVKSECGRTLWTVYTVGERFTYSRVNREYVRKV PTDIIYDALQKAGISVEGDLRDRIAESDGDTLKSIFYAFKYALDMR VENREEDYIQSPVKNASGEFFCSKNAGKSLPQDSDANGAYNIALK GILQLRMLSEQYDPNAESIRLPLITNKAWLTFMQSGMKTWKN

In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with LbCas12a): K538A, K538D, K538E, Y542A, Y542D, Y542E, or K595A, K595D, K595E relative to the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with AsCas12a): K548A, K548D, K548E, N552A, N552D, N552E, or K607A, K607D, K607 relative to the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with CtCas12a): K534A, K534D, K534E, Y538A, Y538D, Y538E, or R591A, R591D, R591E relative to the amino acid sequence of SEQ ID NO: 3.

In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with EeCas12a): K542A, K541D, K541E, N545A, N545D, N545E or K601A, K601D, K601E relative to the amino acid sequence of SEQ ID NO: 4.

In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with Mb3Cas12a): K579A, K579D, K579E, N583A, N583D, N583E or K635A, K635D, K635E relative to the amino acid sequence of SEQ ID NO: 5.

In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with FnCas12a): K613A, K613D, K613E, N617A, N617D, N617E or K671A, K671D, K671E relative to the amino acid sequence of SEQ ID NO: 6.

In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with FnoCas12a): K613A, K613D, K613E, N617A, N617D, N617E or N671A, N671D, N671E relative to the amino acid sequence of SEQ ID NO: 7.

In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with FbCas12a): K617A, K617D, K617E, N621A, N621D, N621E or K678A, K678D, K678E relative to the amino acid sequence of SEQ ID NO: 8.

In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with Lb4Cas12a): K541A, K541D, K541E, N545A, N545D, N545E or K601A, K601D, K601E relative to the amino acid sequence of SEQ ID NO: 9.

In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with MbCas12a): K569A, K569D, K569E, N573A, N573D, N573E or K625A, K625D, K625E relative to the amino acid sequence of SEQ ID NO: 10.

In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with Pb2Cas12a): K562A, K562D, K562E, N566A, N566D, N566E or K619A, K619D, K619E relative to the amino acid sequence of SEQ ID NO: 11.

In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with PgCas12a): K645A, K645D, K645E, N649A, N649D, N649E or K732A, K732D, K732E relative to the amino acid sequence of SEQ ID NO: 12.

In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with AaCas12a): K548A, K548D, K548E, N552A, N552D, N552E or K607A, K607D, K607E relative to the amino acid sequence of SEQ ID NO: 13.

In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with BoCas12a): K592A, K592D, K592E, N596A, N596D, N596E or K653A, K653D, K653E relative to the amino acid sequence of SEQ ID NO: 14.

In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with CMaCas12a): K521A, K521D, K521E, K525A, K525D, K525E or K577A, K577D, K577E relative to the amino acid sequence of SEQ ID NO: 15.

The mutations described herein may be described in the context of a natural Cas12a (any one of SEQ ID NOs: 15) sequence and mutational positions can be carried out by aligning the amino acid sequence of a Cas12a nucleic acid-guided nuclease with, for example, SEQ ID NO: 1 and making the equivalent modification (e.g., substitution) at the equivalent position. By way of example, Table 8 illustrates the equivalent amino acid positions of fifteen orthologous Cas12a nucleic acid-guided nucleases (SEQ ID NOs: 1-15). Any one of the amino acids indicated in Table 8 may be mutated (i.e., via a comparable amino acid substitution).

TABLE 8 Equivalent amino acid positions in homologous Cas12a nucleic acid-guided nuclease WT SEQ ID NO Cas 12a Ortholog AA position AA position AA position AA position SEQ ID NO: 1 LbCas12a G532 K538 Y542 K595 SEQ ID NO: 2 AsCas12a S542 K548 N552 K607 SEQ ID NO: 3 CtCas12a N528 K534 Y538 R591 SEQ ID NO: 4 EeCas12a N535 K541 N545 K601 SEQ ID NO: 5 Mb3Cas12a N573 K579 N583 K635 SEQ ID NO: 6 FnCas12a N607 K613 N617 K671 SEQ ID NO: 7 FnoCas12a N607 K613 N617 N671 SEQ ID NO: 8 FbCas12a N611 K617 N621 K678 SEQ ID NO: 9 Lb4Cas12a N535 K541 N545 K601 SEQ ID NO: 10 MbCas12a N563 K569 N573 K625 SEQ ID NO: 11 Pb2Cas12a G556 K562 N566 K619 SEQ ID NO: 12 PgCas12a D639 K645 N649 K732 SEQ ID NO: 13 AaCas12a S542 K548 N552 K607 SEQ ID NO: 14 BoCas12a K586 K592 N596 K653 SEQ ID NO: 15 CMaCas12a D515 K521 N525 K577

The variant single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 1-15 (excluding the residues listed in Table 8) and contain any conservative mutation one or more residues indicated in Tables 9-13.

It should be appreciated that any of the amino acid mutations described herein, (e.g., K595A) from a first amino acid residue (e.g., K, an amino acid with a basic side chain) to a second amino acid residue (e.g., A, an amino acid with an aliphatic side chain) may also include mutations from the first amino acid residue, lysine, to an amino acid residue that is similar to (e.g., conserved) the second amino acid residue, alanine, such as valine or glycine. As another example, mutation of an amino acid with a positively charged side chain (e.g., arginine, histidine, or lysine) may be a mutation to a second amino acid with an acidic side chain (e.g., glutamic acid or aspartic acid). As another example, mutation of an amino acid with a polar side chain (e.g., serine, threonine, asparagine, or glutamine) may be a mutation to a second amino acid with a positively charged side chain (e.g., arginine, histidine, or lysine). The skilled artisan would recognize that such conservative amino acid substitutions will likely have minor effects on protein structure and are likely to be well tolerated without compromising function. That is, a mutation from one amino acid to a threonine may be an amino acid mutation to a serine; a mutation from one amino acid to an arginine may be an amino acid mutation to a lysine; a mutation from one amino acid to an isoleucine, may be an amino acid mutation to an alanine, valine, methionine, or leucine; a mutation from one amino acid to a lysine may be an amino acid mutation to an arginine; a mutation from one amino acid to an aspartic acid may be an amino acid mutation to a glutamic acid or asparagine; a mutation from one amino acid to a valine may be an amino acid mutation to an alanine, isoleucine, methionine, or leucine; a mutation from one amino acid to a glycine may be an amino acid mutation to an alanine. It should be appreciated, however, that additional conserved amino acid residues would be recognized by the skilled artisan and any of the amino acid mutations to other conserved amino acid residues are also within the scope of this disclosure.

Exemplary variant Cas12a orthologs are shown in tables 9-13.

TABLE 9 Exemplary Variant Ortholog Cas12a’s SEQ ID NO: Variant LbCas12a (in relation to wt LbCas12a SEQ ID NO:1) SEQ ID NO: Variant AsCas12a (in relation to wt AsCas12a SEQ ID NO:2) SEQ ID NO: Variant CtCas12a (in relation to wt CtCas12a SEQ ID NO:3) 16 K595A 55 K607A 94 R591A 17 K595D 56 K607D 95 R591D 18 K595E 57 K607E 96 R591E 19 K538A/K595A 58 K548A/K607A 97 K534A/R591A 20 K538A/K595D 59 K548A/K607D 98 K534A/R591D 21 K538A/K595E 60 K548A/K607E 99 K534A/R591E 22 K538D/K595A 61 K548D/K607A 100 K534D/R591A 23 K538D/K595D 62 K548D/K607D 101 K534D/R591D 24 K538D/K595E 63 K548D/K607E 102 K534D/R591E 25 K538E/K595A 64 K548E/K607A 103 K534E/R591A 26 K538E/K595D 65 K548E/K607D 104 K534E/R591D 27 K538E/K595E 66 K548E/K607E 105 K534E/R591E 28 K538A/Y542A/K595A 67 K548A/N552A/K607A 106 K534A/Y538A/R591A 29 K538A/Y542D/K595A 68 K548A/N552D/K607A 107 K534A/Y538D/R591A 30 K538A/Y542E/K595A 69 K548A/N552E/K607A 108 K534A/Y538E/R591A 31 K538A/Y542A/K595D 70 K548A/N552A/K607D 109 K534A/Y538A/R591D 32 K538A/Y542D/K595D 71 K548A/N552D/K607D 110 K534A/Y538D/R591D 33 K538A/Y542E/K595D 72 K548A/N552E/K607D 111 K534A/Y538E/R591D 34 K538A/Y542A/K595E 73 K548A/N552A/K607E 112 K534A/Y538A/R591E 35 K538A/Y542D/K595E 74 K548A/N552D/K607E 113 K534A/Y538D/R591E 36 K538A/Y542E/K595E 75 K548A/N552E/K607E 114 K534A/Y538E/R591E 37 K538D/Y542A/K595A 76 K548D/N552A/K607A 115 K534D/Y538A/R591A 38 K538D/Y542D/K595A 77 K548D/N552D/K607A 116 K534D/Y538D/R591A 39 K538D/Y542E/K595A 78 K548D/N552E/K607A 117 K534D/Y538E/R591A 40 K538D/Y542A/K595D 79 K548D/N552A/K607D 118 K534D/Y538A/R591D 41 K538D/Y542D/K595D 80 K548D/N552D/K607D 119 K534D/Y538D/R591D 42 K538D/Y542E/K595D 81 K548D/N552E/K607D 120 K534D/Y538E/R591D 43 K538D/Y542A/K595E 82 K548D/N552A/K607E 121 K534D/Y538A/R591E 44 K538D/Y542D/K595E 83 K548D/N552D/K607E 122 K534D/Y538D/R591E 45 K538D/Y542E/K595E 84 K548D/N552E/K607E 123 K534D/Y538E/R591E 46 K538E/Y542A/K595A 85 K548E/N552A/K607A 124 K534E/Y538A/R591A 47 K538E/Y542D/K595A 86 K548E/N552D/K607A 125 K534E/Y538D/R591A 48 K538E/Y542E/K595A 87 K548E/N552E/K607A 126 K534E/Y538E/R591A 49 K538E/Y542A/K595E 88 K548E/N552A/K607D 127 K534E/Y538A/R591D 50 K538E/Y542D/K595E 89 K548E/N552D/K607D 128 K534E/Y538D/R591D 51 K538E/Y542E/K595E 90 K548E/N552E/K607D 129 K534E/Y538E/R591D 52 K538E/Y542A/K595E 91 K548E/N552A/K607E 130 K534E/Y538A/R591E 53 K538E/Y542D/K595E 92 K548E/N552D/K607E 131 K534E/Y538D/R591E 54 K538E/Y542E/K595E 93 K548E/N552E/K607E 132 K534E/Y538E/R591E

TABLE 10 Exemplary Variant Ortholog Cas12a’s SEQ ID NO: Variant EeCas12a (in relation to wt EeCas12a SEQ ID NO:4) SEQ ID NO: Variant Mb3Cas12a (in relation to wt Mb3Cas12a SEQ ID NO:5) SEQ ID NO: Variant FnCas12a (in relation to wt FnCas12a SEQ ID NO:6) 133 K601A 172 K635A 211 K671A 134 K601D 173 K635D 212 K671D 135 K601E 174 K635E 213 K671E 136 K541A/K601A 175 K579A/K635A 214 K613A/K671A 137 K541A/K601D 176 K579A/K635D 215 K613A/K671D 138 K541A/K601E 177 K579A/K635E 216 K613A/K671E 139 K541D/K601A 178 K579D/K635A 217 K613D/K671A 140 K541D/K601D 179 K579D/K635D 218 K613D/K671D 141 K541D/K601E 180 K579D/K635E 219 K613D/K671E 142 K541E/K601A 181 K579E/K635A 220 K613E/K671A 143 K541E/K601D 182 K579E/K635D 221 K613E/K671D 144 K541E/K601E 183 K579E/K635E 222 K613E/K671E 145 K541A/N545A/K601A 184 K579A/N583A/K635A 223 K613A/N617A/K671A 146 K541A/N545D/K601A 185 K579A/N583D/K635A 224 K613A/N617D/K671A 147 K541A/N545E/K601A 186 K579A/N583E/K635A 225 K613A/N617E/K671A 148 K541A/N545A/K601D 187 K579A/N583A/K635D 226 K613A/N617A/K671D 149 K541A/N545D/K601D 188 K579A/N583D/K635D 227 K613A/N617D/K671D 150 K541A/N545E/K601D 189 K579A/N583E/K635D 228 K613A/N617E/K671D 151 K541A/N545A/K601E 190 K579A/N583A/K635E 229 K613A/N617A/K671E 152 K541A/N545D/K601E 191 K579A/N583D/K635E 230 K613A/N617D/K671E 153 K541A/N545E/K601E 192 K579A/N583E/K635E 231 K613A/N617E/K671E 154 K541D/N545A/K601A 193 K579D/N583A/K635A 232 K613D/N617A/K671A 155 K541D/N545D/K601A 194 K579D/N583D/K635A 233 K613D/N617D/K671A 156 K541D/N545E/K601A 195 K579D/N583E/K635A 234 K613D/N617E/K671A 157 K541D/N545A/K601D 196 K579D/N583A/K635D 235 K613D/N617A/K671D 158 K541D/N545D/K601D 197 K579D/N583D/K635D 236 K613D/N617D/K671D 159 K541D/N545E/K601D 198 K579D/N583E/K635D 237 K613D/N617E/K671D 160 K541D/N545A/K601E 199 K579D/N583A/K635E 238 K613D/N617A/K671E 161 K541D/N545D/K601E 200 K579D/N583D/K635E 239 K613D/N617D/K671E 162 K541D/N545E/K601E 201 K579D/N583E/K635E 240 K613D/N617E/K671E 163 K541E/N545A/K601A 202 K579E/N583A/K635A 241 K613E/N617A/K671A 164 K541E/N545D/K601A 203 K579E/N583D/K635A 242 K613E/N617D/K671A 165 K541E/N545E/K601A 204 K579E/N583E/K635A 243 K613E/N617E/K671A 166 K541E/N545A/K601D 205 K579E/N583A/K635D 244 K613E/N617A/K671D 167 K541E/N545D/K601D 206 K579E/N583D/K635D 245 K613E/N617D/K671D 168 K541E/N545E/K601D 207 K579E/N583E/K635D 246 K613E/N617E/K671D 169 K541E/N545A/K601E 208 K579E/N583A/K635E 247 K613E/N617A/K671E 170 K541E/N545D/K601E 209 K579E/N583D/K635E 248 K613E/N617D/K671E 171 K541E/N545E/K601E 210 K579E/N583E/K635E 249 K613E/N617E/K671E

TABLE 11 Exemplary Variant Ortholog Cas12a’s SEQ ID NO: Variant FnoCas12a (in relation to wt FnoCas12a SEQ ID NO:7) SEQ ID NO: Variant FbCas12a (in relation to wt FbCas12a SEQ ID NO:8) SEQ ID NO: Variant Lb4as12a (in relation to wt Lb4Cas12a SEQ ID NO:9) 250 N671A 289 K678A 328 K601A 251 N671D 290 K678D 329 K601D 252 N671E 291 K678E 330 K601E 253 K613A/N671A 292 K617A/K678A 331 K541A/K601A 254 K613A/N671D 293 K617A/K678D 332 K541A/K601D 255 K613A/N671E 294 K617A/K678E 333 K541A/K601E 256 K613D/N671A 295 K617D/K678A 334 K541D/K601A 257 K613D/N671D 296 K617D/K678D 335 K541D/K601D 258 K613D/N671E 297 K617D/K678E 336 K541D/K601E 259 K613E/N671A 298 K617E/K678A 337 K541E/K601A 260 K613E/N671D 299 K617E/K678D 338 K541E/K601D 261 K613E/N671E 300 K617E/K678E 339 K541E/K601E 262 K613A/N617A/N671A 301 K617A/N621A/K678A 340 K541A/ N545A/ K601A 263 K613A/N617D/N671A 302 K617A/N621D/K678A 341 K541A/ N545D/ K601A 264 K613A/N617E/N671A 303 K617A/N621E/K678A 342 K541A/ N545E/ K601A 265 K613A/N617A/N671D 304 K617A/N621A/K678D 343 K541A/ N545A/ K601D 266 K613A/N617D/N671D 305 K617A/N621D/K678D 344 K541A/ N545D/ K601D 267 K613A/N617E/N671D 306 K617A/N621E/K678D 345 K541A/ N545E/ K601D 268 K613A/N617A/N671E 307 K617A/N621A/K678E 346 K541A/ N545A/ K601E 269 K613A/N617D/N671E 308 K617A/N621D/K678E 347 K541A/ N545D/ K601E 270 K613A/N617E/N671E 309 K617A/N621E/K678E 348 K541A/ N545E/ K601E 271 K613D/N617A/N671A 310 K617D/N621A/K678A 349 K541D/ N545A/ K601A 272 K613D/N617D/N671A 311 K617D/N621D/K678A 350 K541D/ N545D/ K601A 273 K613D/N617E/N671A 312 K617D/N621E/K678A 351 K541D/ N545E/ K601A 274 K613D/N617A/N671D 313 K617D/N621A/K678D 352 K541D/ N545A/ K601D 275 K613D/N617D/N671D 314 K617D/N621D/K678D 353 K541D/ N545D/ K601D 276 K613D/N617E/N671D 315 K617D/N621E/K678D 354 K541D/ N545E/ K601D 277 K613D/N617A/N671E 316 K617D/N621A/K678E 355 K541D/ N545A/ K601E 278 K613D/N617D/N671E 317 K617D/N621D/K678E 356 K541D/ N545D/ K601E 279 K613D/N617E/N671E 318 K617D/N621E/K678E 357 K541D/ N545E/ K601E 280 K613E/N617A/N671A 319 K617E/N621A/K678A 358 K541E/ N545A/ K601A 281 K613E/N617D/N671A 320 K617E/N621D/K678A 359 K541E/ N545D/ K601A 282 K613E/N617E/N671A 321 K617E/N621E/K678A 360 K541E/ N545E/ K601A 283 K613E/N617A/N671D 322 K617E/N621A/K678D 361 K541E/ N545A/ K601D 284 K613E/N617D/N671D 323 K617E/N621D/K678D 362 K541E/ N545D/ K601D 285 K613E/N617E/N671D 324 K617E/N621E/K678D 363 K541E/ N545E/ K601D 286 K613E/N617A/N671E 325 K617E/N621A/K678E 364 K541E/ N545A/ K601E 287 K613E/N617D/N671E 326 K617E/N621D/K678E 365 K541E/ N545D/ K601E 288 K613E/N617E/N671E 327 K617E/N621E/K678E 366 K541E/ N545E/ K601E

TABLE 12 Exemplary Variant Ortholog Cas12a’s SEQ ID NO: Variant MbCas12a (in relation to wt MbCas12a SEQ ID NO:10) SEQ ID NO: Variant Pb2Cas12a (in relation to wt Pb2Cas12a SEQ ID NO:11) SEQ ID NO: Variant PgCas12a (in relation to wt PgCas12a SEQ ID NO:12) 367 K625A 406 K619A 445 K732A 368 K625D 407 K619D 446 K732D 369 K625E 408 K619E 447 K732E 370 K569A/K625A 409 K562A/K619A 448 K645A/K732A 371 K569A/K625D 410 K562A/K619D 449 K645A/K732D 372 K569A/K625E 411 K562A/K619E 450 K645A/K732E 373 K569D/K625A 412 K562D/K619A 451 K645D/K732A 374 K569D/K625D 413 K562D/K619D 452 K645D/K732D 375 K569D/K625E 414 K562D/K619E 453 K645D/K732E 376 K569E/K625A 415 K562E/K619A 454 K645E/K732A 377 K569E/K625D 416 K562E/K619D 455 K645E/K732D 378 K569E/K625E 417 K562E/K619E 456 K645E/K732E 379 K569A/ N573A/ K625A 418 K562A/N566A/K619A 457 K645A/N649A/K732A 380 K569A/ N573D/ K625A 419 K562A/N566D/K619A 458 K645A/N649D/K732A 381 K569A/ N573E/ K625A 420 K562A/N566E/K619A 459 K645A/N649E/K732A 382 K569A/ N573A/ K625D 421 K562A/N566A/K619D 460 K645A/N649A/K732D 383 K569A/ N573D/ K625D 422 K562A/N566D/K619D 461 K645A/N649D/K732D 384 K569A/ N573E/ K625D 423 K562A/N566E/K619D 462 K645A/N649E/K732D 385 K569A/ N573A/ K625E 424 K562A/N566A/K619E 463 K645A/N649A/K732E 386 K569A/ N573D/ K625E 425 K562A/N566D/K619E 464 K645A/N649D/K732E 387 K569A/ N573E/ K625E 426 K562A/N566E/K619E 465 K645A/N649E/K732E 388 K569D/ N573A/ K625A 427 K562D/N566A/K619A 466 K645D/N649A/K732A 389 K569D/ N573D/ K625A 428 K562D/N566D/K619A 467 K645D/N649D/K732A 390 K569D/ N573E/ K625A 429 K562D/N566E/K619A 468 K645D/N649E/K732A 391 K569D/ N573A/ K625D 430 K562D/N566A/K619D 469 K645D/N649A/K732D 392 K569D/ N573D/ K625D 431 K562D/N566D/K619D 470 K645D/N649D/K732D 393 K569D/ N573E/ K625D 432 K562D/N566E/K619D 471 K645D/N649E/K732D 394 K569D/ N573A/ K625E 433 K562D/N566A/K619E 472 K645D/N649A/K732E 395 K569D/ N573D/ K625E 434 K562D/N566D/K619E 473 K645D/N649D/K732E 396 K569D/ N573E/ K625E 435 K562D/N566E/K619E 474 K645D/N649E/K732E 397 K569E/ N573A/ K625A 436 K562E/N566A/K619A 475 K645E/N649A/K732A 398 K569E/ N573D/ K625A 437 K562E/N566D/K619A 476 K645E/N649D/K732A 399 K569E/ N573E/ K625A 438 K562E/N566E/K619A 477 K645E/N649E/K732A 400 K569E/ N573A/ K625D 439 K562E/N566A/K619D 478 K645E/N649A/K732D 401 K569E/ N573D/ K625D 440 K562E/N566D/K619D 479 K645E/N649D/K732D 402 K569E/ N573E/ K625D 441 K562E/N566E/K619D 480 K645E/N649E/K732D 403 K569E/ N573A/ K625E 442 K562E/N566A/K619E 481 K645E/N649A/K732E 404 K569E/ N573D/ K625E 443 K562E/N566D/K619E 482 K645E/N649D/K732E 405 K569E/ N573E/ K625E 444 K562E/N566E/K619E 483 K645E/N649E/K732E

TABLE 13 Exemplary Variant Ortholog Cas12a’s SEQ ID NO: Variant AaCas12a (in relation to wt AaCas12a SEQ ID NO: 13) SEQ ID NO: Variant BoCas12a (in relation to wt BoCas12a SEQ ID NO:14) SEQ ID NO: Variant CMaCas12a (in relation to wt CMaCas12a SEQ ID NO:15) 484 K607A 523 K653A 562 K577A 485 K607D 524 K653D 563 K577D 486 K607E 525 K653E 564 K577E 487 K548A/K607A 526 K592A/K653A 565 K521A/K577A 488 K548A/K607D 527 K592A/K653D 566 K521A/K577D 489 K548A/K607E 528 K592A/K653E 567 K521A/K577E 490 K548D/K607A 529 K592D/K653A 568 K521D/K577A 491 K548D/K607D 530 K592D/K653D 569 K521D/K577D 492 K548D/K607E 531 K592D/K653E 570 K521D/K577E 493 K548E/K607A 532 K592E/K653A 571 K521E/K577A 494 K548E/K607D 533 K592E/K653D 572 K521E/K577D 495 K548E/K607E 534 K592E/K653E 573 K521E/K577E 496 K548A/N552A/K607A 535 K592A/N596A/K653A 574 K521A/N525A/K577A 497 K548A/N552D/K607A 536 K592A/N596D/K653A 575 K521A/N525D/K577A 498 K548A/N552E/K607A 537 K592A/N596E/K653A 576 K521A/N525E/K577A 499 K548A/N552A/K607D 538 K592A/N596A/K653D 577 K521A/N525A/K577D 500 K548A/N552D/K607D 539 K592A/N596D/K653D 578 K521A/N525D/K577D 501 K548A/N552E/K607D 540 K592A/N596E/K653D 579 K521A/N525E/K577D 502 K548A/N552A/K607E 541 K592A/N596A/K653E 580 K521A/N525A/K577E 503 K548A/N552D/K607E 542 K592A/N596D/K653E 581 K521A/N525D/K577E 504 K548A/N552E/K607E 543 K592A/N596E/K653E 582 K521A/N525E/K577E 505 K548D/N552A/K607A 544 K592D/N596A/K653A 583 K521D/N525A/K577A 506 K548D/N552D/K607A 545 K592D/N596D/K653A 584 K521D/N525D/K577A 507 K548D/N552E/K607A 546 K592D/N596E/K653A 585 K521D/N525E/K577A 508 K548D/N552A/K607D 547 K592D/N596A/K653D 586 K521D/N525A/K577D 509 K548D/N552D/K607D 548 K592D/N596D/K653D 587 K521D/N525D/K577D 510 K548D/N552E/K607D 549 K592D/N596E/K653D 588 K521D/N525E/K577D 511 K548D/N552A/K607E 550 K592D/N596A/K653E 589 K521D/N525A/K577E 512 K548D/N552D/K607E 551 K592D/N596D/K653E 590 K521D/N525D/K577E 513 K548D/N552E/K607E 552 K592D/N596E/K653E 591 K521D/N525E/K577E 514 K548E/N552A/K607A 553 K592E/N596A/K653A 592 K521E/N525A/K577A 515 K548E/N552D/K607A 554 K592E/N596D/K653A 593 K521E/N525D/K577A 516 K548E/N552E/K607A 555 K592E/N596E/K653A 594 K521E/N525E/K577A 517 K548E/N552A/K607D 556 K592E/N596A/K653D 595 K521E/N525A/K577D 518 K548E/N552D/K607D 557 K592E/N596D/K653D 596 K521E/N525D/K577D 519 K548E/N552E/K607D 558 K592E/N596E/K653D 597 K521E/N525E/K577D 520 K548E/N552A/K607E 559 K592E/N596A/K653E 598 K521E/N525A/K577E 521 K548E/N552D/K607E 560 K592E/N596D/K653E 599 K521E/N525D/K577E 522 K548E/N552E/K607E 561 K592E/N596E/K653E 600 K521E/N525E/K577E

In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least 70% identical to any one of SEQ ID NOs: 16-600. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least 75% identical to any one of SEQ ID NOs: 16-600 16-600. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least 80% identical to any one of SEQ ID NOs: 16-600. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least 85% identical to any one of SEQ ID NOs: 16-600. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least 90% identical to any one of SEQ ID NOs: 16-600. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least 95% identical to any one of SEQ ID NOs: 16-600. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 16-600. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is any one of SEQ ID NOs: 16-600.

The mutations described herein are described in the context of the WT LbCas12a (e.g., SEQ ID NO: 1) sequence and mutational positions can be carried out by aligning the amino acid sequence of a Cas12a nucleic acid-guided nuclease with SEQ ID NO: 1 and making the equivalent modification (e.g., substitution) at the equivalent position. By way of example, the mutations described herein may be applied to a Cas12a enzyme shown in Table 7, or any other homolog Cas 12a thereof by aligning the amino acid sequence of the Cas 12a to SEQ ID NO: 1 and making the modifications described in Tables 9-13 (changes to the wildtype residue to alanine, aspartic acid or glutamic acid or conservative equivalents at the Cas12a ortholog’s equivalent position (e.g., see Table 8 for an example of equivalent residue positions).

For example, in addition to the variant LbCas12a sequences in Table 9 (variant sequences SEQ ID Nos: 16-54), like variants are envisioned for AsCas12a (variant sequences SEQ ID Nos: 55-93), CtCas12a (variant sequences SEQ ID Nos: 94-132), EeCas12a (variant sequences SEQ ID Nos: 133-171), Mb3Cas12a (variant sequences SEQ ID Nos: 172-210), FnCas12a (variant sequences SEQ ID Nos: 211-249), FnoCas12a (variant sequences SEQ ID Nos: 250-288), FbCas12a (variant sequences SEQ ID Nos: 289-327), Lb4Cas12a (variant sequences SEQ ID Nos: 328-366), MbCas12a (variant sequences SEQ ID Nos: 367-405), Pb2Cas12a (variant sequences SEQ ID Nos: 406-444), PgCas12a (variant sequences SEQ ID Nos: 445-483), AaCas12a (variant sequences SEQ ID Nos: 484-522), BoCas12a (variant sequences SEQ ID Nos: 523-561), and CmaCas12a (variant sequences SEQ ID Nos: 562-600). In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least 70% identical to any one of SEQ ID NOs: 16-600 and contains an amino acid substitution(s) listed in Tables 9-13 or the equivalent in a different ortholog. In some embodiments, the single-strand-specific Cas 12a nucleic acid-guided nuclease is at least 75% identical to any one of SEQ ID NOs: 16-600 and contains an amino acid substitution(s) listed in Tables 9-13 or the equivalent in a different ortholog. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least 80% identical to any one of SEQ ID NOs: 16-600 and contains an amino acid substitution(s) listed in Tables 9-13 or the equivalent in a different ortholog. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least 85% identical to any one of SEQ ID NOs: 16-600 and contains an amino acid substitution(s) listed in Tables 9-13 or the equivalent in a different ortholog. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least 90% identical to any one of SEQ ID NOs: 16-600 and contains an amino acid substitution(s) listed in Tables 9-13 or the equivalent in a different ortholog. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least 95% identical to any one of SEQ ID NOs: 16-600 and contains an amino acid substitution(s) listed in Tables 9-13 or the equivalent in a different ortholog. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least %, 97%, 98% or 99% identical to any one of SEQ ID NOs: 16-600 and contains an amino acid substitution(s) listed in Tables 9-13 or the equivalent in a different ortholog. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is any one of SEQ ID NOs: 16-600.

The single-strand-specific Cas12a nucleic acid-guided nucleases described herein may be any Cas12a nucleic acid-guided nuclease that largely prevents double-stranded nucleic acid unwinding and R-loop formation. The single-strand-specific Cas12a nucleic acid-guided nucleases described herein may also be any Cas12a nucleic acid-guided nuclease that lacks cis-cleavage activity yet maintains trans-nucleic acid-guided nuclease activity on single-stranded nucleic acid molecules. Such single-strand-specific Cas 12a nucleic acid-guided nucleases may be generated via the mutations described herein.

Additionally, or alternatively, such single-strand-specific Cas12a nucleic acid-guided nucleases may be generated via post-translational modifications (e.g., acetylation). The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be an acetylated Cas12a enzyme. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be an LbCas12a (i.e., SEQ ID NO: 1) with an acetylated K595 (K595K^(Ac)) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be an AsCas12a (i.e., SEQ ID NO: 2) with an acetylated K607 (K607K^(Ac)) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be a CtCas12a (i.e., SEQ ID NO: 3) with an acetylated R591 (R591R^(Ac)) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be an EeCas12a (i.e., SEQ ID NO: 4) with an acetylated K601 (K607K^(Ac)) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be an Mb3Cas12a (i.e., SEQ ID NO: 5) with an acetylated K635 (K635K^(Ac)) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be an FnCas12a (i.e., SEQ ID NO: 6) with an acetylated K671 (K671K^(Ac)) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be an FnoCas12a (i.e., SEQ ID NO: 7) with an acetylated N671 (N671K^(Ac)) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be an FbCas12a (i.e., SEQ ID NO: 8) with an acetylated K678 (K678K^(Ac)) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be an Lb4Cas12a (i.e., SEQ ID NO: 9) with an acetylated K601 (K601K^(Ac)) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be an MbCas12a (i.e., SEQ ID NO: 10) with an acetylated K625 (K625K^(Ac)) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be a Pb2Cas12a (i.e., SEQ ID NO: 11) with an acetylated K619 (K619K^(Ac)) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be a PgCas12a (i.e., SEQ ID NO: 12) with an acetylated K732 (K732K^(Ac)) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be an AaCas12a (i.e., SEQ ID NO: 13) with an acetylated K607 (K607K^(Ac)) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be an BoCas12a (i.e., SEQ ID NO: 14) with an acetylated K653 (K653K^(Ac)) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be an CmaCas12a (i.e., SEQ ID NO: 15) with an acetylated K577 (K577K^(Ac)) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be a Cas12a ortholog acetylated at the amino acid of the ortholog equivalent to K595 of SEQ ID NO: 1. Acetylation of Cas 12a can be carried out with any suitable acetyltransferase. For a discussion and methods for disabling of Cas12a by ArVA5, see Dong, et al., Nature Structural and Molecular Bio., 26(4):308-14 (2019). For example, LbCas12a can be incubated with AcrVA5 in order to acetylate the K595 residue, thereby deactivating the dsDNA activity (e.g., FIG. 7 ). In addition to acetylation, phosphorylation and methylation of select amino acid residues may be employed.

Bulky Modifications

In addition to the modalities of adjusting the ratio of the concentration of the blocked nucleic acid molecules to the concentration of the RNP2 and altering the domains of the variant nucleic acid-guided nuclease of RNP2 that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules to vary dsDNA vs. ssDNA recognition properties as described in detail above, the present disclosure additionally contemplates use of “bulky modifications” at the 5′ and/or 3′ ends and/or at internal nucleic acid bases of the blocked nucleic acid molecule and/or using modifications between internal nucleic acid bases. FIG. 8A is an illustration of the steric hindrance at the PAM-interacting (PI) domain in a nucleic acid-guided nuclease caused by 5′ and 3′ modifications to a blocked nucleic acid molecule. At top in FIG. 8A is an illustration of the target stand and non-target strand, and below this is an illustration of a self-hybridized blocked nucleic acid molecule comprising three loop regions, as well as bulky modifications on the 5′ and 3′ ends of the blocked nucleic acid molecule. Example “bulky modifications” include a fluorophore and quencher pair (as shown here) or biotin, but in general encompass molecules with a size of about 1 nm or less, or 0.9 nm or less, or 0.8 nm or less, or 0.7 nm or less, or 0.6 nm or less, or 0.5 nm or less, or 0.4 nm or less, or 0.3 nm or less, or 0.2 nm or less, or 0.1 nm or less, or 0.05 nm or less, or as small as 0.025 nm or less.

In the illustration at center, the blocked nucleic acid molecule with the 5′ and 3′ ends comprising a fluorophore and a quencher is shown being cleaved at the loop regions. Note that the bulky modifications in this embodiment also allow the blocked nucleic acid molecule to act as a reporter moiety; that is, when the loop regions of the blocked nucleic acid molecule are cleaved, the short nucleotide segments of the non-target strand dehybridize from the target strand due to low T_(m), thereby separating the fluorophore and quencher such that fluorescence from the fluorophore is no longer quenched and can be detected. In the illustration at bottom, the intact blocked nucleic acid molecule with the bulky modifications (at left) sterically hinders interaction with the PAM-interacting (PI) domain of the nucleic acid-guided nuclease in RNP2 such that the intact blocked nucleic acid molecule cannot be cleaved via cis-cleavage by the nucleic acid-guided nuclease. However, once the loop regions of the blocked nucleic acid molecule are cleaved (via, e.g., trans-cleavage from RNP1 (at right)) and the short nucleotide segments of the non-target strand dehybridize from the target strand, leaving the 3′ end of the now single-stranded target strand is now free to initiate R-loop formation with RNP2. R-loop formation leads to cis-cleavage of the single-strand target strand, and subsequent activation of trans-cleavage of RNP2.

FIG. 8B illustrates five exemplary variations of blocked nucleic acid molecules with bulky modifications, including at the 5′ and/or 3′ ends of a self-hybridizing blocked nucleic acid molecule and/or at internal nucleic acid bases of the blocked nucleic acid molecule. Embodiment (i) illustrates a self-hybridizing blocked nucleic acid molecule having a fluorophore at its 5′ end and a quencher at its 3′ end. Embodiment (ii) illustrates a self-hybridizing blocked nucleic acid molecule having a fluorophore and a quencher at internal nucleic acid bases flanking a loop sequence. Embodiment (iii) illustrates a self-hybridizing blocked nucleic acid molecule having a fluorophore at its 5′ end and a quencher at its 3′ end as well as having a fluorophore and a quencher at internal nucleic acid bases where the internal fluorophore and quencher flank a loop sequence. The fluorophore/quencher embodiments work as long as the fluorophore and quencher are at a distance of about 10-11 nm or less apart. Embodiment (iv) illustrates a self-hybridizing blocked nucleic acid molecule having a biotin molecule at its 5′ end, and embodiment (v) illustrates a self-hybridizing blocked nucleic acid molecule having a biotin at an internal nucleic acid base. Note that bulky modifications of internal nucleic acid bases often are made at or near a loop region of a blocked nucleic acid molecule (or blocked target molecule). The loop regions are regions of the blocked nucleic acid molecules — in addition to the 5′ and 3′ ends — that may be vulnerable to unwinding.

Modifications can be used in self-hybridized blocked nucleic acid molecules lacking a PAM or those comprising a PAM, partially self-hybridized blocked nucleic acid molecules lacking a PAM or those comprising a PAM, or reverse PAM molecules. Other variations include using RNA loops instead of DNA loops if a Cas 13 nucleic acid-guided nuclease is used as the nucleic acid-guided nuclease in RNP1, or entire RNA molecules if a Cas 13 nucleic acid-guided nuclease is used as the nucleic acid-guided nuclease in RNP1 and RNP2.

FIGS. 8C, 8D and 8E list exemplary bulky modifications for 5′, 3′, and internal positions in blocked nucleic acid molecules, and Table 14 below lists sequences of exemplary self-hybridizing blocked nucleic acid molecules. 56-FAM stands for 5′ 6-FAM (6-fluorescein amidite); and 3BHQ stands for 3′ BLACK HOLE QUENCHER®-1.

TABLE 14 Bulky Modifications N o. SEQ ID NO: Molecule Name Molecule Sequence (5′→3′) 5′ FAM + 3′ BHQ 1 601 5′F_U29_Q /56-FAM/GATCCATTTTATTTTAGATCATATATATACATGATCGG ATC/3BHQ_1/ 2 602 5′F_1C armor_U29 _Q /56-FAM/CGATCCATTTTATTTTAGATCATATATATACATGATCG GATCG/3BHQ_1/ 3 603 5′F_2CC armor_U29 _Q /56-FAM/CCGATCCATTTTATTTTAGATCATATATATACATGATC GGATCGG/3BHQ_1/ 4 604 5′F_1A armor_U29 _Q /56-FAM/AGATCCATTTTATTTTAGATCATATATATACATGATCG GATCT/3BHQ_1/ 5 605 5′F_2AT armor_U29 _Q /56-FAM/ATGATCCATTTTATTTTAGATCATATATATACATGATC GGATCAT/3BHQ_1/ 6 606 5′F_U250_ Q /56-FAM/GATATATAAAAAAAAAAAGATCATATACATATATGAT CATATATC/3BHQ_1/ 7 607 5′F_1C armor_U25 0_Q /56-FAM/CGATATATAAAAAAAAAAAGATCATATACATATATGA TCATATATCG/3BHQ_1/ 8 608 5′F_2CC armor_U25 0_Q /56-FAM/CCGATATATAAAAAAAAAAAGATCATATACATATATG ATCATATATCGG/3BHQ_1/ 9 609 5′F_1A armor_U25 0_Q /56-FAM/AGATATATAAAAAAAAAAAGATCATATACATATATGA TCATATATCT/3BHQ_1/ 10 610 5′F_2AT armor_U25 0_Q /56-FAM/ATGATATATAAAAAAAAAAAGATCATATACATATATG ATCATATATCAT/3BHQ_1/ 5’Fluorsceine (modification on base) + 3’ BHQ 11 611 5′FdT_U29 _Q /5FluorT/GATCCATTTTATTTTAGATCATATATATACATGATC GGATCA/3BHQ_1/ 12 612 5′FdT_1C armor_U29 _Q /SFluorT/CGATCCATTTTATTTTAGATCATATATATACATGAT CGGATCGA/3BHQ_1/ 13 605 5′FdT_1A armor_U29 _Q A/iFluorT/GATCCATTTTATTTTAGATCATATATATACATGAT CGGATCAT/3BHQ_1/ 14 613 5′FdT_U25 0_Q /5FluorT/GATATATAAAAAAAAAAAGATCATATACATATATG ATCATATATCA/3BHQ_1/ 15 614 5′FdT_1C armor_U25 0_Q /5FluorT/CGATATATAAAAAAAAAAAGATCATATACATATAT GATCATATATCGA/3BHQ_1/ 16 610 5′FdT_1A armor_U25 0_Q A/iFluorT/GATATATAAAAAAAAAAAGATCATATACATATAT GATCATATATCAT/3BHQ_1/ 5’FAM + Internal Fluorsceine (modification on base) + 3’ BHQ 17 601 5′F_IntFdt_ U29_Q /56-FAM/GA/iFluorT/CCATTTTATTTTAGATCATATATATACATG ATCGGATC/3BHQ_1/ 18 606 5′F_IntFdt_ U250_Q /56-FAM/GA/iFluorT/ATATAAAAAAAAAAAGATCATATACATAT ATGATCATATATC/3BHQ_1/ 19 602 5′F_1C armor_IntF dt_U29_Q /56-FAM/CGA/iFluorT/CCATTTTATTTTAGATCATATATATACAT GATCGGATCG/3BHQ_1/ 20 604 5′F_1A armor_IntF dt_U29_Q /56-FAM/AGA/iFluorT/CCATTTTATTTTAGATCATATATATACAT GATCGGATCT/3BHQ_1/ 21 607 5′F_1C armor_IntF dt_U250_Q /56-FAM/CGA/iFluorT/ATATAAAAAAAAAAAGATCATATACATA TATGATCATATATCG/3BHQ_1/ 22 609 5′F_1A armor_IntF dt_U250_Q /56-FAM/AGA/iFluorT/ATATAAAAAAAAAAAGATCATATACATA TATGATCATATATCT/3BHQ_1/ 23 603 5′F_2CC armor_IntF dt_U29_Q /56-FAM/CCGA/iFluorT/CCATTTTATTTTAGATCATATATATACA TGATCGGATCGG/3BHQ_1/ 24 605 5′F_2AT armor_IntF dt_U29_Q /56-FAM/ATGA/iFluorT/CCATTTTATTTTAGATCATATATATACA TGATCGGATCAT/3BHQ_1/ 25 608 5′F_2CC armor_IntF dt_U250_Q /56-FAM/CCGA/iFluorT/ATATAAAAAAAAAAAGATCATATACAT ATATGATCATATATCGG/3BHQ_1/ 26 610 5′F_2AT armor_IntF dt_U250_Q /56-FAM/ATGA/iFluorT/ATATAAAAAAAAAAAGATCATATACAT ATATGATCATATATCAT/3BHQ_1/

Applications of the Cascade Assay

The present disclosure describes cascade assays for detecting a target nucleic acid of interest in a sample that provide instantaneous or nearly instantaneous results even at ambient temperatures at 16° C. and above, allow for massive multiplexing and minimum workflow, yet provide accurate results at low cost. Moreover, the various embodiments of the cascade assay are notable in that, with the exception of the gRNA in RNP1, the cascade assay components stay the same no matter what target nucleic acid(s) of interest are being detected and RNP1 is easily reprogrammed. Moreover, the cascade assay can be massively multiplexed for detecting several to many to target nucleic acid molecules simultaneously. For example, the assay may be designed to detect one to several to many different pathogens (e.g., testing for many different pathogens in one assay), or the assay may be designed to detect one to several to many different sequences from the same pathogen (e.g., to increase specificity and sensitivity), or a combination of the two.

As described above, early and accurate identification of, e.g., infectious agents, microbe contamination, and variant nucleic acid sequences that indicate the present of such diseases such as cancer or contamination by heterologous sources is important in order to select correct therapeutic treatment, identify tainted food, pharmaceuticals, cosmetics and other commercial goods; and to monitor the environment. The cascade assay described herein can be applied in diagnostics for, e.g., infectious disease (including but not limited to Covid, HIV, flu, the common cold, Lyme disease, STDs, chicken pox, diptheria, mononucleosis, hepatitis, UTIs, pneumonia, tetanus, rabies, malaria, dengue fever, Ebola, plague; see Table 1), for rapid liquid biopsies and companion diagnostics (biomarkers for cancers, early detection, progression, monitoring; see Table 4), prenatal testing (including but not limited to chromosomal abnormalities and genetic diseases such as sickle cell, including over-the-counter versions of prenatal testing assays), rare disease testing (achondroplasia, Addison’s disease, α1-antitrypsin deficiency, multiple sclerosis, muscular dystrophy, cystic fibrosis, blood factor deficiencies), SNP detection/DNA profiling/epigenetics, genotyping, low abundance transcript detection, labeling for cell or droplet sorting, in situ nucleic acid detection, sample prep, library quantification of NGS, screening biologics (including engineered therapeutic cells for genetic integrity and/or contamination), development of agricultural products, food compliance testing and quality control (e.g., detection of genetically modified products, confirmation of source for high value commodities, contamination detection), infectious disease in livestock, infectious disease in cash crops, livestock breeding, drug screening, personal genome testing including clinical trial stratification, personalized medicine, nutrigenomics, drug development and drug therapy efficacy, transplant compatibility and monitoring, environmental testing and forensics, and bioterrorism agent monitoring.

Target nucleic acids of interest are derived from samples as described in more detail above. Suitable samples for testing include, but are not limited to, any environmental sample, such as air, water, soil, surface, food, clinical sites and products, industrial sites and products, pharmaceuticals, medical devices, nutraceuticals, cosmetics, personal care products, agricultural equipment and sites, and commercial samples, and any biological sample obtained from an organism or a part thereof, such as a plant, animal, or microbe. In some embodiments, the biological sample is obtained from an animal subject, such as a human subject. A biological sample may be any solid or fluid sample obtained from, excreted by or secreted by any living organism, including, without limitation, single celled organisms, such as bacteria, yeast, protozoans, and amoebas among others, multicellular organisms including plants or animals, including samples from a healthy or apparently healthy human subject or a human patient affected by a condition or disease to be diagnosed or investigated, such as an infection with a pathogenic microorganism, such as a pathogenic bacteria or virus.

For example, a biological sample can be a biological fluid obtained from a human or non-human (e.g., livestock, pets, wildlife) animal, and may include but is not limited to blood, plasma, serum, urine, stool, sputum, mucous, lymph fluid, synovial fluid, bile, ascites, pleural effusion, seroma, saliva, cerebrospinal fluid, aqueous or vitreous humor, or any bodily secretion, a transudate, an exudate (for example, fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint (for example, a normal joint or a joint affected by disease, such as rheumatoid arthritis, osteoarthritis, gout or septic arthritis), or a swab of skin or mucosal membrane surface (e.g., a nasal or buccal swab).

In some embodiments, the sample can be a viral or bacterial sample or a biological sample that has been minimally processed, e.g., only treated with a brief lysis step prior to detection. In other embodiments, minimal processing can include thermal lysis at an elevated temperature to release nucleic acids. Suitable methods are contemplated in USPN 9,493,736, among other references. Common methods for cell lysis involve thermal, chemical, enzymatic, or mechanical treatment of the sample or a combination of those (see, e.g., Example I below). In some embodiments, minimal processing can include treating the sample with chaotropic salts such as guanidine isothiocyanate or guanidine HCl. Suitable methods are contemplated in USPN 8,809,519 and USPN 7,893,251, among other references. In some embodiments, minimal processing may include contacting the sample with reducing agents such as DTT or TCEP and EDTA to inactivate inhibitors and/or other nucleases present in the crude samples. In other embodiments, minimal processing for biofluids may include centrifuging the samples to obtain cell-debris free supernatant before applying the reagents. Suitable methods are contemplated in USPN 8,809,519, among other references. In still other embodiments, minimal processing may include performing DNA/RNA extraction to get purified nucleic acids before applying CRISPR Cascade reagents.

Table 15 below lists exemplary commercial sample processing kits, and Table 16 below lists point of care processing techniques.

TABLE 15 Exemplary Commercial Sample and Nucleic Acid Processing Kits Manufacturer Kit Sample Type Output Lysing and extraction methods Qiagen® DNeasy™ Blood & Tissue Kits small volumes of blood genomic DNA Isolation of Genomic DNA from Small Volumes of Blood dried blood spots 1. Uses Chemical and Biological/Enzymatic lysis methods urine 2. Uses SPE with Column Purification tissues laser- microdissected tissues Isolation of Genomic DNA from Tissues 1.Uses Chemical and Biological/Enzymatic lysis methods 2.Used to dissolve and lyse tissue sections completely, higher temperature and longer time incubations up to 24 hours are used Qiagen® QIAamp® UCP Pathogen Mini Handbook whole blood microbial DNA Specific pretreatment protocols are suggested depending on sample type with or without the use of kits for Mechanical Lysis Method before downstream applications. swabs microbial DNA purification cultures -- pelleted microbial cells body fluids Downstream applications contain: 1.Chemical and Biological/Enzymaticlysis methods 2. SPE with Column Purification Qiagen® QIAamp® Viral RNA Kits plasma and serum viral DNA 1.Uses Chemical lysis methods CSF 2.Uses SPE with Column Purification urine other cell-free body fluids cell-culture supernatants swabs Zymo Research™ Quick- DNA™Microprep Kit whole blood genomic DNA 1.Uses chemical lysis methods plasma serum 2. Uses SPE with column purification body fluids buffy coat lymphocytes swabs cultured cells Zymo Research™ Quick-DNA™ Fungal/Bacterial Miniprep Kit A. fumigatus Microbial DNA Uses Bead lysis and pretreatment with: C. albicans N. crassa S. cerevisiae 1.Chemical lysis methods with chaotropic salts S. pombe mycelium Gram positive bacteria 2. NAE with SPE with silica matrices Gram negative bacteria

TABLE 16 Point of Care Sample Processing Techniques Steps Protocol Example 1 Protocol Example 2 Protocol Example 3 Field-deployable viral diagnostics using CRISPR-Cas13 Science, Streamlined inactivation, amplification, and Cas13-based detection of SARS-CoV-2 Lucira Health™ 27;360(6387):444-448 (2018) Nat Commun, 11: 5921 (2020) 1. Cell disruption (lysis) and inactivation of nucleases Samples were thermally treated at -40° C. for -15 minutes for nuclease deactivation, thereafter at 90° C. for 5 minutes for viral deactivation. A NP swab or saliva sample was lysed and inactivated for 10 minutes with thermal treatment. These samples were incubated for 5 min at 40° C., followed by 5 min at 70° C. (or 5 min at 95° C., if saliva) Lucira Health uses a single buffer that lyses and inactivates nucleases and/or inhibitors. In POC setting, cell disruption and inactivation of nucleases is done commonly through thermal lysis. Sample Types: A nasal swab is directly added to a single lysing/reaction buffer and vigorously stirred to release the viral particulates from the swab. Urine Saliva Diluted blood (1:3 with PBS) Targets: Viruses Target: SARS-Cov-2 2. Assay on crude sample This is usually a direct assay on the crude sample post cell disruption and inactivation of nucleases. No extraction is usually performed. Thermally treated biological samples(above) were used directly for amplification and detection of pathogenic nucleic acid. Thermally treated biological samples(above) were used directly for amplification and detection of pathogenic nucleic acid. Processed biological sample is used in an isothermal reaction for pathogenic nucleic acid detection.

FIG. 9 shows a lateral flow assay (LFA) device that can be used to detect the cleavage and separation of a signal from a reporter moiety. For example, the reporter moiety may be a single-stranded or double-stranded oligonucleotide with terminal biotin and fluorescein amidite (FAM) modifications; and, as described above, the reporter moiety may also be part of a blocked nucleic acid. The LFA device may include a pad with binding particles, such as gold nanoparticles functionalized with anti-FAM antibodies; a control line with a first binding moiety attached, such as avidin or streptavidin; a test line with a second binding moiety attached, such as antibodies; and an absorption pad. After completion of a cascade assay (see FIGS. 2A, 3A, and 3B), the assay reaction mix is added to the pad containing the binding particles, (e.g., antibody labeled gold nanoparticles). When the target nucleic acid of interest is present, a reporter moiety is cleaved, and when the target nucleic acid of interest is absent, the reporter is not cleaved.

A moiety on the reporter binds to the binding particles and is transported to the control line. When the target nucleic acid of interest is absent, the reporter moiety is not cleaved, and the first binding moiety binds to the reporter moiety, with the binding particles attached. When the target nucleic acid of interest is present, one portion of the cleaved reporter moiety binds to the first binding moiety, and another portion of the cleaved reporter moiety bound to the binding particles via the moiety binds to the second binding moiety. In one example, anti-FAM gold nanoparticles bind to a FAM terminus of a reporter moiety and flow sequentially toward the control line and then to the test line. For reporters that are not trans-cleaved, gold nanoparticles attach to the control line via biotin-streptavidin and result in a dark control line. In a negative test, since the reporter has not been cleaved, all gold conjugates are trapped on control line due to attachment via biotin-streptavidin. A negative test will result in a dark control line with a blank test line. In a positive test, reporter moieties have been trans-cleaved by the cascade assay, thereby separating the biotin terminus from the FAM terminus. For cleaved reporter moieties, nanoparticles are captured at the test line due to anti-FAM antibodies. This positive test results in a dark test line in addition to a dark control line.

The components of the cascade assay may be provided in various kits for testing at, e.g., point of care facilities, in the field, pandemic testing sites, and the like. In one aspect, the kit for detecting a target nucleic acid of interest in a sample includes: first ribonucleoprotein complexes (RNP1s), second ribonucleoprotein complexes (RNP2s), blocked nucleic acid molecules, and reporter moieties. The first complex (RNP1) comprises a first nucleic acid-guided nuclease and a first gRNA, where the first gRNA includes a sequence complementary to the target nucleic acid(s) of interest. Binding of the first complex (RNP1) to the target nucleic acid(s) of interest activates trans-cleavage activity of the first nucleic acid-guided nuclease. The second complex (RNP2) comprises a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest. The blocked nucleic acid molecule comprises a sequence complementary to the second gRNA, where trans-cleavage of the blocked nucleic acid molecule results in an unblocked nucleic acid molecule and the unblocked nucleic acid molecule can bind to the second complex (RNP2), thereby activating the trans-cleavage activity of the second nucleic acid-guided nuclease. Activating trans-cleavage activity in RNP2 results in an exponential increase in unblocked nucleic acid molecules and in active reporter moieties, where reporter moieties are nucleic acid molecules and/or are operably linked to the blocked nucleic acid molecules and produce a detectable signal upon cleavage by RNP2.

In a second aspect, the kit for detecting a target nucleic acid molecule in sample includes: first ribonucleoprotein complexes (RNP1s), second ribonucleoprotein complexes (RNP2s), template molecules, blocked primer molecules, a polymerase, NTPs, and reporter moieties. The first ribonucleoprotein complex (RNP1) comprises a first nucleic acid-guided nuclease and a first gRNA, where the first gRNA includes a sequence complementary to the target nucleic acid of interest and where binding of RNP1 to the target nucleic acid(s) of interest activates trans-cleavage activity of the first nucleic acid-guided nuclease. The second complex (RNP2) comprises a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest. The template molecules comprise a primer binding domain (PBD) sequence as well as a sequence corresponding to a spacer sequence of the second gRNA. The blocked primer molecules comprise a sequence that is complementary to the PBD on the template nucleic acid molecule and a blocking moiety.

Upon binding to the target nucleic acid of interest, RNP1 becomes active triggering trans-cleavage activity that cuts at least one of the blocked primer molecules to produce at least one unblocked primer molecule. The unblocked primer molecule hybridizes to the PBD of one of the template nucleic acid molecules, is trimmed of excess nucleotides by the 3′-to-5′ exonuclease activity of the polymerase and is then extended by the polymerase and NTPs to form a synthesized activating molecule with a sequence that is complementary to the second gRNA of RNP2 (i.e., the synthesized activating molecule is the target strand). Upon activating RNP2, additional trans-cleavage activity is initiated, cleaving at least one additional blocked primer molecule. Continued cleavage of blocked primer molecules and subsequent activation of more RNP2s proceeds at an exponential rate. A signal is generated upon cleavage of a reporter molecule by active RNP2 complexes; therefore, a change in signal production indicates the presence of the target nucleic acid molecule.

Any of the kits described herein may further include a sample collection device, e.g., a syringe, lancet, nasal swab, or buccal swab for collecting a biological sample from a subject, and/or a sample preparation reagent, e.g., a lysis reagent. Each component of the kit may be in separate container or two or more components may be in the same container. The kit may further include a lateral flow device used for contacting the biological sample with the reaction mixture, where a signal is generated to indicate the presence or absence of the target nucleic acid molecule of interest. In addition, the kit may further include instructions for use and other information.

EXAMPLES

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent or imply that the experiments below are all of or the only experiments performed. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific aspects without departing from the spirit or scope of the invention as broadly described. The present aspects are, therefore, to be considered in all respects as illustrative and not restrictive.

Example I: Preparation of Nucleic Acids of Interest

Mechanical lysis: Nucleic acids of interest may be isolated by various methods depending on the cell type and source (e.g., tissue, blood, saliva, environmental sample, etc.). Mechanical lysis is a widely used cell lysis method and may be used to extract nucleic acids from bacterial, yeast, plant and mammalian cells. Cells are disrupted by agitating a cell suspension with “beads” at high speeds (beads for disrupting various types of cells can be sourced from, e.g., OPS Diagnostics (Lebanon NJ, US) and MP Biomedicals (Irvine, CA, USA)). Mechanical lysis via beads begins with harvesting cells in a tissue or liquid, where the cells are first centrifuged and pelleted. The supernatant is removed and replaced with a buffer containing detergents as well as lysozyme and protease. The cell suspension is mixed to promote breakdown of the proteins in the cells and the cell suspension then is combined with small beads (e.g., glass, steel, or ceramic beads) that are mixed (e.g., vortexed) with the cell suspension at high speeds. The beads collide with the cells, breaking open the cell membrane with shear forces. After “bead beating”, the cell suspension is centrifuged to pellet the cellular debris and beads, and the supernatant may be purified via a nucleic acid binding column (such as the MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit from ThermoFisher (Waltham, MA, USA) and others from Qiagen (Hilden, Germany), TakaraBio (San Jose, CA, USA), and Biocomma (Shenzen, China)) to collect the nucleic acids (see the discussion of solid phase extraction below).

Solid phase extraction (SPE): Another method for capturing nucleic acids is through solid phase extraction. SPE involves a liquid and stationary phase, which selectively separates the target analyte (here, nucleic acids) from the liquid in which the cells are suspended based on specific hydrophobic, polar, and/or ionic properties of the target analyte in the liquid and the stationary solid matrix. Silica binding columns and their derivatives are the most commonly used SPE techniques, having a high binding affinity for DNA under alkaline conditions and increased salt concentration; thus, a highly alkaline and concentrated salt buffer is used. The nucleic acid sample is centrifuged through a column with a highly porous and high surface area silica matrix, where binding occurs via the affinity between negatively charged nucleic acids and positively charged silica material. The nucleic acids bind to the silica matrices, while the other cell components and chemicals pass through the matrix without binding. One or more wash steps typically are performed after the initial sample binding (i.e., the nucleic acids to the matrix), to further purify the bound nucleic acids, removing excess chemicals and cellular components non-specifically bound to the silica matrix. Alternative versions of SPE include reverse SPE and ion exchange SPE, and use of glass particles, cellulose matrices, and magnetic beads.

Thermal lysis: Thermal lysis involves heating a sample of mammalian cells, virions, or bacterial cells at high temperatures thereby damaging the cellular membranes by denaturizing the membrane proteins. Denaturizing the membrane proteins results in the release of intracellular DNA. Cells are generally heated above 90° C., however time and temperature may vary depending on sample volume and sample type. Once lysed, typically one or more downstream methods, such as use of nucleic acid binding columns for solid phase extraction as described above, are required to further purify the nucleic acids.

Physical lysis: Common physical lysis methods include sonication and osmotic shock. Sonication involves creating and rupturing of cavities or bubbles to release shockwaves, thereby disintegrating the cellular membranes of the cells. In the sonication process, cells are added into lysis buffer, often containing phenylmethylsulfonyl fluoride, to inhibit proteases. The cell samples are then placed in a water bath and a sonication wand is placed directly into the sample solution. Sonication typically occurs between 20-50 kHz, causing cavities to be formed throughout the solution as a result of the ultrasonic vibrations; subsequent reduction of pressure then causes the collapse of the cavity or bubble resulting in a large amount of mechanical energy being released in the form of a shockwave that propagates through the solution and disintegrates the cellular membrane. The duration of the sonication pulses and number of pulses performed varies depending on cell type and the downstream application. After sonication, the cell suspension typically is centrifuged to pellet the cellular debris and the supernatant containing the nucleic acids may be further purified by solid phase extraction as described above.

Another form of physical lysis is osmotic shock, which is most typically used with mammalian cells. Osmotic shock involves placing cells in DI/distilled water with no salt added. Because the salt concentration is lower in the solution than in the cells, water is forced into the cell causing the cell to burst, thereby rupturing the cellular membrane. The sample is typically purified and extracted by techniques such as e.g., solid phase extraction or other techniques known to those of skill in the art.

Chemical lysis: Chemical lysis involves rupturing cellular and nuclear membranes by disrupting the hydrophobic-hydrophilic interactions in the membrane bilayers via detergents. Salts and buffers (such as, e.g., Tris-HCl pH8) are used to stabilize pH during extraction, and chelating agents (such as ethylenediaminetetraacetic acid (EDTA)) and inhibitors (e.g., Proteinase K) are also added to preserve the integrity of the nucleic acids and protect against degradation. Often, chemical lysis is used with enzymatic disruption methods (see below) for lysing bacterial cell walls. In addition, detergents are used to lyse and break down cellular membranes by solubilizing the lipids and membrane proteins on the surface of cells. The contents of the cells include, in addition to the desired nucleic acids, inner cellular proteins and cellular debris. Enzymes and other inhibitors are added after lysis to inactivate nucleases that may degrade the nucleic acids. Proteinase K is commonly added after lysis, destroying DNase and RNase enzymes capable of degrading the nucleic acids. After treatment with enzymes, the sample is centrifuged, pelleting cellular debris, while the nucleic acids remain in the solution. The nucleic acids may be further purified as described above.

Another form of chemical lysis is the widely used procedure of phenol-chloroform extraction. Phenol-chloroform extraction involves the ability for nucleic acids to remain soluble in an aqueous solution in an acidic environment, while the proteins and cellular debris can be pelleted down via centrifugation. Phenol and chloroform ensure a clear separation of the aqueous and organic (debris) phases. For DNA, a pH of 7-8 is used, and for RNA, a more acidic pH of 4.5 is used.

Enzymatic lysis: Enzymatic disruption methods are commonly combined with other lysis methods such as those described above to disrupt cellular walls (bacteria and plants) and membranes. Enzymes such as lysozyme, lysostaphin, zymolase, and protease are often used in combination with other techniques such as physical and chemical lysis. For example, one can use cellulase to disrupt plant cell walls, lysosomes to disrupt bacterial cell walls and zymolase to disrupt yeast cell walls.

Example II: RNP Formation

For RNP complex formation, 250 nM of LbCas12a nuclease protein was incubated with 375 nM of a target specific gRNA in 1X Buffer (10 mM Tris-HCl, 100 µg/mL BSA) with 2-15 mM MgCl₂ at 25° C. for 20 minutes. The total reaction volume was 2 µL. Other ratios of LbCas12a nuclease to gRNAs were tested, including 1:1, 1:2 and 1:5. The incubation temperature ranged from 16° C. - 37° C., and the incubation time ranged from 10 minutes to 4 hours.

Example III: Blocked Nucleic Acid Molecule Formation

Ramp cooling: For formation of the secondary structure of blocked nucleic acid molecules, 2.5 µM of a blocked nucleic acid molecule (any of Formulas I - IV) was mixed in a T50 buffer (20 mM Tris HCl, 50 mM NaCl) with 10 mM MgCl₂ for a total volume of 50 µL. The reaction was heated to 95° C. at 1.6° C./second and incubated at 95° C. for 5 minutes to dehybridize any secondary structures. Thereafter, the reaction was cooled to 37° C. at 0.015° C./second to form the desired secondary structure.

Snap cooling: For formation of the secondary structure of blocked nucleic acid molecules, 2.5 µM of a blocked nucleic acid molecule (any of Formulas I - IV) was mixed in a T50 buffer (20 mM Tris HCl, 50 mM NaCl) with 10 mM MgCl₂ for a total volume of 50 µL. The reaction was heated to 95° C. at 1.6° C./second and incubated at 95° C. for 5 minutes to dehybridize any secondary structures. Thereafter, the reaction was cooled to room temperature by removing the heat source to form the desired secondary structure.

Snap cooling on ice: For formation of the secondary structure of blocked nucleic acid molecules, 2.5 µM of a blocked nucleic acid molecule (any of Formulas I -IV) was mixed in a T50 buffer (20 mM Tris HCl, 50 mM NaCl) with 10 mM MgCl₂ for a total volume of 50 µL. The reaction was heated to 95° C. at 1.6° C./second and incubated at 95° C. for 5 minutes to dehybridize any secondary structures. Thereafter, the reaction was cooled to room temperature by placing the reaction tube on ice to form the desired secondary structure.

Example IV: Reporter Moiety Formation

The reporter moieties used in the reactions herein were single-stranded DNA oligonucleotides 5-9 bases in length (e.g., with sequences of TTATT, TTTATTT, ATTAT, ATTTATTTA, AAAAA, or AAAAAAAAA) with a fluorophore and a quencher attached on the 5′ and 3′ ends, respectively. In one example using a Cas12a cascade, the fluorophore was FAM-6 and the quencher was IOWA BLACK® (Integrated DNA Technologies, Coralville, IA). In another example using a Cas13 cascade, the reporter moieties were single-stranded RNA oligonucleotides 5-10 bases in length (e.g., r(U)n, r(UUAUU)n, r(A)n).

Example V: Cascade Assay

Format I (final reaction mix components added at the same time): RNP1 was assembled using the LbCas12a nuclease and a gRNA for the Methicillin resistant Staphylococcus aureus (MRSA) DNA according to the RNP complex formation protocol described in Example II (for this sequence, see Example VI). Briefly, 250 nM LbCas12a nuclease was assembled with 375 nM of the MRSA-target specific gRNA. Next, RNP2 was formed using the LbCas12a nuclease and a gRNA specific for a selected blocked nucleic acid molecule (Formula I - IV) using 500 nM LbCas12a nuclease assembled with 750 nM of the blocked nucleic acid-specific gRNA incubated in 1X NEB 2.1 Buffer (New England Biolabs, Ipswich, MA) with 5 mM MgCl₂ at 25° C. for 20-40 minutes. Following incubation, RNP1s were diluted to a concentration of 75 nM LbCas12a: 112.5 nM gRNA. Thereafter, the final reaction was carried out in 1X Buffer, with 500 nM of the ssDNA reporter moiety, 1X ROX dye (Thermo Fisher Scientific, Waltham, MA) for passive reference, 2.5 mM MgCl₂, 4 mM NaCl, 15 nM LbCas12a: 22.5 nM gRNA RNP1, 20 nM LbCas12a: 35 nM gRNA RNP2, and 50 nM blocked nucleic acid molecule (any one of Formula I - IV) in a total volume of 9 µL. 1 µL of MRSA DNA target (with samples having as low as three copies and as many as 30000 copies - see FIGS. 6-14 ) was added to make a final volume of 10 µL. The final reaction was incubated in a thermocycler at 25° C. with fluorescence measurements taken every 1 minute.

Format II (RNP1 and MRSA target pre-incubated before addition to final reaction mix): RNP1 was assembled using the LbCas12a nuclease and a gRNA for the MRSA DNA according to RNP formation protocol described in Example II (for this sequence, see Example VI). Briefly, 250 nM LbCas12a nuclease was assembled with 375 nM of the MRSA-target specific gRNA. Next, RNP2 was formed using the LbCas12a nuclease and a gRNA specific for a selected blocked nucleic acid molecule (Formula I -IV) using 500 nM LbCas12a nuclease assembled with 750 nM of the blocked nucleic acid-specific gRNA incubated in 1X NEB 2.1 Buffer (New England Biolabs, Ipswich, MA) with 5 mM MgCl₂ at 25° C. for 20-40 minutes. Following incubation, RNPls were diluted to a concentration of 75 nM LbCas12a: 112.5 nM gRNA. After dilution, the formed RNP1 was mixed with 1µL of MRSA DNA target and incubated at 16° C. - 37° C. for up to 10 minutes to activate RNP1. The final reaction was carried out in 1X Buffer, with 500 nM of the ssDNA reporter moiety, 1X ROX dye (Thermo Fisher Scientific, Waltham, MA) for passive reference, 2.5 mM MgCl₂, 4 mM NaCl, the pre-incubated and activated RNP1, 20 nM LbCas12a: 35 nM gRNA RNP2, and 50 nM blocked nucleic acid molecule (any one of Formula I - IV) in a total volume of 9µL. The final reaction was incubated in a thermocycler at 25° C. with fluorescence measurements taken every 1 minute.

Format III (RNP1 and MRSA target pre-incubated before addition to final reaction mix and blocked nucleic acid molecule added to final reaction mix last): RNP1 was assembled using the LbCas12a nuclease and a gRNA for the MRSA DNA according to the RNP complex formation protocol described in Example II (for this sequence, see Example VI). Briefly, 250 nM LbCas12a nuclease was assembled with 375 nM of the MRSA-target specific gRNA. Next, RNP2 was formed using the LbCas12a nuclease and a gRNA specific for a selected blocked nucleic acid molecule (Formula I - IV) using 500 nM LbCas12a nuclease assembled with 750 nM of the blocked nucleic acid-specific gRNA incubated in 1X NEB 2.1 Buffer (New England Biolabs, Ipswich, MA) with 5 mM MgCl₂ at 25° C. for 20-40 minutes. Following incubation, RNPls were diluted to a concentration of 75 nM LbCas12a: 112.5 nM gRNA. After dilution, the formed RNP1 was mixed with 1 µL of MRSA DNA target and incubated at 16° C. - 37° C. for up to 10 minutes to activate RNP1. The final reaction was carried out in 1X Buffer, with 500 nM of the ssDNA reporter moiety, 1X ROX dye (Thermo Fisher Scientific, Waltham, MA) for passive reference, 2.5 mM MgCl₂, 4 mM NaCl, the pre-incubated and activated RNP1, and 20 nM LbCas12a: 35 nM gRNA RNP2 in a total volume of 9 µL. Once the reaction mix was made, 1 µL (50 nM) blocked nucleic acid molecule (any one of Formula I - IV) was added for a total volume of 10 µL. The final reaction was incubated in a thermocycler at 25° C. with fluorescence measurements taken every 1 minute.

Example VI: Detection of MRSA and Test Reaction Conditions

To detect the presence of Methicillin resistant Staphylococcus aureus (MRSA) and determine the sensitivity of detection with the cascade assay, titration experiments with a MRSA DNA target nucleic acid of interest were performed. The MRSA DNA sequence (NCBI Reference Sequence NC: 007793.1) is as follows. SEQ ID NO: 615:

ATGAAAAAGATAAAAATTGTTCCACTTATTTTAATAGTTGTAGTTGTCGGGTTTGGTATATATTTTTATG CTTCAAAAGATAAAGAAATTAATAATACTATTGATGCAATTGAAGATAAAAATTTCAAACAAGTTTATAA AGATAGCAGTTATATTTCTAAAAGCGATAATGGTGAAGTAGAAATGACTGAACGTCCGATAAAAATATAT AATAGTTTAGGCGTTAAAGATATAAACATTCAGGATCGTAAAATAAAAAAAGTATCTAAAAATAAAAAAC GAGTAGATGCTCAATATAAAATTAAAACAAACTACGGTAACATTGATCGCAACGTTCAATTTAATTTTGT TAAAGAAGATGGTATGTGGAAGTTAGATTGGGATCATAGCGTCATTATTCCAGGAATGCAGAAAGACCAA AGCATACATATTGAAAATTTAAAATCAGAACGTGGTAAAATTTTAGACCGAAACAATGTGGAATTGGCCA ATACAGGAACAGCATATGAGATAGGCATCGTTCCAAAGAATGTATCTAAAAAAGATTATAAAGCAATCGC TAAAGAACTAAGTATTTCTGAAGACTATATCAAACAACAAATGGATCAAAATTGGGTACAAGATGATACC TTCGTTCCACTTAAAACCGTTAAAAAAATGGATGAATATTTAAGTGATTTCGCAAAAAAATTTCATCTTA CAACTAATGAAACAGAAAGTCGTAACTATCCTCTAGGAAAAGCGACTTCACATCTATTAGGTTATGTTGG TCCCATTAACTCTGAAGAATTAAAACAAAAAGAATATAAAGGCTATAAAGATGATGCAGTTATTGGTAAA AAGGGACTCGAAAAACTTTACGATAAAAAGCTCCAACATGAAGATGGCTATCGTGTCACAATCGTTGACG ATAATAGCAATACAATCGCACATACATTAATAGAGAAAAAGAAAAAAGATGGCAAAGATATTCAACTAAC TATTGATGCTAAAGTTCAAAAGAGTATTTATAACAACATGAAAAATGATTATGGCTCAGGTACTGCTATC CACCCTCAAACAGGTGAATTATTAGCACTTGTAAGCACACCTTCATATGACGTCTATCCATTTATGTATG GCATGAGTAACGAAGAATATAATAAATTAACCGAAGATAAAAAAGAACCTCTGCTCAACAAGTTCCAGAT TACAACTTCACCAGGTTCAACTCAAAAAATATTAACAGCAATGATTGGGTTAAATAACAAAACATTAGAC GATAAAACAAGTTATAAAATCGATGGTAAAGGTTGGCAAAAAGATAAATCTTGGGGTGGTTACAACGTTA CAAGATATGAAGTGGTAAATGGTAATATCGACTTAAAACAAGCAATAGAATCATCAGATAACATTTTCTT TGCTAGAGTAGCACTCGAATTAGGCAGTAAGAAATTTGAAAAAGGCATGAAAAAACTAGGTGTTGGTGAA GATATACCAAGTGATTATCCATTTTATAATGCTCAAATTTCAAACAAAAATTTAGATAATGAAATATTAT TAGCTGATTCAGGTTACGGACAAGGTGAAATACTGATTAACCCAGTACAGATCCTTTCAATCTATAGCGC ATTAGAAAATAATGGCAATATTAACGCACCTCACTTATTAAAAGACACGAAAAACAAAGTTTGGAAGAAA AATATTATTTCCAAAGAAAATATCAATCTATTAACTGATGGTATGCAACAAGTCGTAAATAAAACACATA AAGAAGATATTTATAGATCTTATGCAAACTTAATTGGCAAATCCGGTACTGCAGAACTCAAAATGAAACA AGGAGAAACTGGCAGACAAATTGGGTGGTTTATATCATATGATAAAGATAATCCAAACATGATGATGGCT ATTAATGTTAAAGATGTACAAGATAAAGGAATGGCTAGCTACAATGCCAAAATCTCAGGTAAAGTGTATG ATGAGCTATATGAGAACGGTAATAAAAAATACGATATAGATGAATAA

Briefly, a RNP1 was preassembled with a gRNA sequence designed to target MRSA DNA. Specifically, RNP1 was designed to target a 20 bp region of the mecA gene of MRSA: TGTATGGCATGAGTAACGAA (SEQ ID NO: 616). An RNP2 was preassembled with a gRNA sequence designed to target the unblocked nucleic acid molecule that results from unblocking (i.e., linearizing) blocked nucleic acid molecule U29 (FIG. 10A). The reaction mix contained the preassembled RNP1, preassembled RNP2, and a blocked nucleic acid molecule, in a buffer (pH of about 8) containing 4 mM MgCl₂ and 101 mM NaCl.

FIG. 10A shows the structure and segment parameters of molecule U29. Note molecule U29 has a secondary structure free energy value of -5.84 kcal/mol and relatively short self-hybridizing, double-stranded regions of 5 bases and 6 bases. FIGS. 10B - 10H show the results achieved for detection of 3E4 copies, 30 copies, 3 copies and 0 copies of the mecA gene of MRSA (n = 3) at 25° C. with varying concentrations of blocked nucleic acid, RNP2 and reporter moiety. FIG. 10B shows the results achieved when 100 nM blocked nucleic acid molecules, 10 nM RNP2s and 500 nM reporter moieties are used. Thus, in this experiment, the ratio of blocked nucleic acid molecules to RNP2s is 10:1. Note first that with 3E4 copies, nearly 100% of the reporters are cleaved at t = 0 with a signal-to-noise ratio of 28.06 at 0 minutes, a signal-to-noise ratio of 24.23 at 5 minutes, and a signal-to-noise ratio of 21.01 at 10 minutes. Additionally, the signal-to-noise ratios for detection with 30 copies of MRSA target is 12.45 at 0 minutes, 14.07 at 5 minutes and 16.16 at 10 minutes; and the signal-to-noise ratios for detection with 3 copies of MRSA target is 1.79 at 0 minutes, 1.64 at 5 minutes and is 2.04 at 10 minutes. Note the measured fluorescence at 0 copies increases only slightly over the 10- and 30-minutes intervals, resulting in a flat negative. A flat negative (the results obtained over the time period for 0 copies) demonstrates that there is very little non-specific or undesired signal generation in the system. Note that the negative when the ratio of blocked nucleic acid molecules to RNP2s is 10:1 is flatter than those in FIGS. 10C through 10H.

FIG. 10C shows the results achieved when 50 nM blocked nucleic acid molecules, 10 nM RNP2s and 500 nM reporter moieties are used. Thus, in this experiment, the ratio of blocked nucleic acid molecules to RNP2s is 5:1. Note first that with 3E4 copies, again nearly 100% of the reporters are cleaved at t = 0 with a signal-to-noise ratio of 12.85, a signal-to-noise ratio of 10.51 at 5 minutes, and a signal-to-noise ratio of 8.18 at 10 minutes. Additionally, the signal-to-noise ratios for detection with 30 copies of MRSA target is 5.85 at 0 minutes, 6.44 at 5 minutes and 6.48 at 10 minutes; and the signal-to-noise ratios for detection with 3 copies of MRSA target is 1.54 at 0 minutes, 1.61 at 5 minutes and is 1.71 at 10 minutes. Note the measured fluorescence at 0 copies increases, resulting in less of a flat negative than the 10:1 ratio of blocked nucleic acid molecules to RNP2.

FIG. 10D shows the results achieved when 50 nM blocked nucleic acid molecules, 10 nM RNP2s and 2500 nM reporter moieties are used. Thus, in this experiment, the ratio of blocked nucleic acid molecules to RNP2s is 5:1. With 3E4 copies, again nearly 100% of the reporters are cleaved at t = 0 with a signal-to-noise ratio of 34.92, a signal-to-noise ratio of 30.62 at 5 minutes, and a signal-to-noise ratio of 25.81 at 10 minutes. Additionally, the signal-to-noise ratios for detection with 30 copies of MRSA target is 7.97 at 0 minutes, 1.73 at 5 minutes and 10.50 at 10 minutes; and the signal-to-noise ratios for detection with 3 copies of MRSA target is 1.65 at 0 minutes, 1.73 at 5 minutes and is 1.82 at 10 minutes. Note the measured fluorescence at 0 copies increases, resulting in less of a flat negative than the 10:1 ratio of blocked nucleic acid molecules to RNP2s, but likely due to the 5x increase in the concentration of reporter moieties; however, note also that a higher concentration of reporter moieties allows for a higher signal-to-noise ratio for 3E4 and 30 copies of MRSA target.

FIG. 10E shows the results achieved when 100 nM blocked nucleic acid molecules, 20 nM RNP2s and 500 nM reporter moieties are used and 4 mM NaCl. Thus, in this experiment, the ratio of blocked nucleic acid molecules to RNP2s is 5:1 but double the concentration of both of these molecules than that shown in FIGS. 10C and 10D. With 3E4 copies, again nearly 100% of the reporters are cleaved at t = 0 with a signal-to-noise ratio of 11.89, a signal-to-noise ratio of 8.97 at 5 minutes, and a signal-to-noise ratio of 6.53 at 10 minutes. Additionally, the signal-to-noise ratios for detection with 30 copies of MRSA target is 5.46 at 0 minutes, 5.85 at 5 minutes and 5.43 at 10 minutes; and the signal-to-noise ratios for detection with 3 copies of MRSA target is 1.58 at 0 minutes, 1.65 at 5 minutes and is 1.80 at 10 minutes. Note the measured fluorescence at 0 copies increases, resulting in less of a flat negative than the 10:1 ratio of blocked nucleic acid molecules to RNP2s shown in FIG. 10B. Note also that the ratio of blocked nucleic acid molecules to RNP2s (5:1) appears to be more important than the ultimate concentration (100 nM/20 nM) by comparison to FIG. 10D where the ratio of blocked nucleic acid molecules to RNP2s was also 5:1 however the concentration of blocked nucleic acid molecules was 50 nM and the concentration of RNP2 was 10 nM.

FIG. 10F shows the results achieved when 50 nM blocked nucleic acid molecules, 20 nM RNP2s and 500 nM reporter moieties are used and using a concentration of 4 mM NaCl. In this experiment the ratio of blocked nucleic acid molecules to RNP2s is 2.5:1. With 3E4 copies, again nearly 100% of the reporters are cleaved at t = 0 with a signal-to-noise ratio of 25.85, a signal-to-noise ratio of 21.36 at 5 minutes, and a signal-to-noise ratio of 16.24 at 10 minutes. Additionally, the signal-to-noise ratios for detection with 30 copies of MRSA target is 5.28 at 0 minutes, 6.19 at 5 minutes and 7.02 at 10 minutes; and the signal-to-noise ratios for detection with 3 copies of MRSA target is very low at 0 minutes, 1.53 at 5 minutes and is 1.73 at 10 minutes. Note the measured fluorescence at 0 copies increases, resulting in less of a flat negative than the 10:1 ratio of blocked nucleic acid molecules to RNP2s shown in FIG. 10B. Note also that the signal-to-noise ratio for all concentrations was reduced at the 2.5:1 ratio of blocked nucleic acid molecules to RNP2s.

FIG. 10G shows the results achieved when 50 nM blocked nucleic acid molecules, 20 nM RNP2s and 500 nM reporter moieties are used and using a concentration of 10 mM NaCl. Thus, in this experiment, the ratio of blocked nucleic acid molecules to RNP2s is 2.5:1. With 3E4 copies, again nearly 100% of the reporters are cleaved at t = 0 with a signal-to-noise ratio of 12.75, a signal-to-noise ratio of 7.78 at 5 minutes, and a signal-to-noise ratio of 3.66 at 10 minutes. Additionally, the signal-to-noise ratios for detection with 30 copies of MRSA target is 6.09 at 0 minutes, 6.23 at 5 minutes and 3.58 at 10 minutes; and the signal-to-noise ratios for detection with 3 copies of MRSA target is very low at 0 minutes, 1.40 at 5 minutes and is 1.62 at 10 minutes. Note the measured fluorescence at 0 copies increases, resulting in less of a flat negative than the 10:1 ratio of blocked nucleic acid molecules to RNP2s shown in FIG. 10B. Note also that the signal-to-noise ratio for all concentrations was reduced substantially at the 2.5:1 ratio of blocked nucleic acid molecules to RNP2s and that the NaCl concentration at 10 mM vs. 4 mM (FIG. 10F) did not make much of a difference.

FIG. 10H shows the results achieved when 100 nM blocked nucleic acid molecules, 20 nM RNP2s and 500 nM reporter moieties are used and using a concentration of 10 mM NaCl. Thus, in this experiment, the ratio of blocked nucleic acid molecules to RNP2s is 5:1. With 3E4 copies, again nearly 100% of the reporters are cleaved at t = 0 with a signal-to-noise ratio of 77.38, a signal-to-noise ratio of 74.18 at 5 minutes, and a signal-to-noise ratio of 67.90 at 10 minutes. Additionally, the signal-to-noise ratios for detection with 30 copies of MRSA target is 5.94 at 0 minutes, 7.45 at 5 minutes and 9.73 at 10 minutes; and the signal-to-noise ratios for detection with 3 copies of MRSA target is 1.66 at 0 minutes, 2.13 at 5 minutes and is 2.38 at 10 minutes. Note the measured fluorescence at 0 copies increases slightly, resulting in less of a flat negative than the 10:1 ratio of blocked nucleic acid molecules to RNP2s shown in FIG. 10B. Note also that the signal-to-noise ratio for all concentrations was increased substantially at the 5:1 ratio of blocked nucleic acid molecules to RNP2s as compared to the 2.5:1 ration of blocked nucleic acid molecules to RNP2s. In summary, the results shown in FIGS. 10B - 10H indicate that a 5:1 ratio of blocked nucleic acid molecules to RNP2s or greater leads to higher signal-to-noise ratios for all concentrations of MRSA target.

Example VII: Homology Modeling and Mutation Structure Analysis

The variant nucleic acid-guided nucleases presented herein were developed in the following manner: For protein engineering and amino acid substitution model predictions, a first Protein Data Bank (pdb) file with the amino acid sequence and structure information for the RNP comprising the base nucleic acid-guided nuclease to be mutated, the gRNA and a bound dsDNA target nucleic acid was obtained. (For structural information for RNPs comprising AsCas12s and LbCas12a, see, e.g., Yamano, et al., Molecular Cell, 67:633-45 (2017).) Desired and/or random amino acid substitutions were then “made” to the base nucleic acid-guided nuclease (LbCas12a)., the resulting structural change to the base nucleic acid-guided nuclease due to each amino acid substitution was used to generate updated files for the resulting RNPs comprising each of the variant nucleic acid-guided nucleases using SWISS-MODEL and the original pdf file as a reference template. SWISS-MODEL worked well in the present case as the amino acid sequences of wildtype LbCas12a was known, as were the planned amino acid substitutions. The output of the updated files for each variant nucleic acid-guided nuclease included a root mean square deviation (RMSD) value for the structural changes compared to the RNP complex for wt LbCas12a in Angstrom units (i.e., a measurement of the difference between the backbones of wt LbCas12a and the variant nucleic acid-guided nuclease) and the updated pdb files of the variant nucleic acid-guided nucleases are further assessed at the point of mutations for changes in the hydrogen bonds compared to the reference original pdb file of the nuclease.

After SWISS modeling, an independent step for calculating free energy was performed using, e.g., a Flex ddG module based on the program Rosetta CM to extract locally destabilizing mutations. This was used as a proxy for amino acid interference with PAM regions of the DNA to assess the probability of unwinding of the target nucleic acid. (See, e.g., Shanthirabalan, et al., Proteins: Structure, Function, and Bioinformatics 86(8):853-867 (2018); and Barlow, et al., J. Physical Chemistry B, 122(21):5389-99 (2018).)

Generally, the results of the SWISS-Model and Rosetta analysis indicated that stable enzyme function related to the PAM domain would require a global RMSD value range from 0.1 to 2.1 angstroms, and the following ΔΔG Flex Values: for stabilizing mutations ΔΔG < -1.0kcal/mol; for neutral mutations: -1.0kcal/mol < ΔΔG < 1.0 kcal/mol; and for destabilizing mutations: ΔΔG ≥ 1.0 kcal/mol. Sixteen single mutations were identified that, singly or in combination, met the calculated criteria. Structural modeling for mutations at four exemplary amino acid residues are described below.

FIG. 6A shows the result of protein structure prediction using Rosetta and SWISS modeling of wildtype LbCas12a (Lachnospriaceae bacterium Cas12a). Protein structure prediction using Rossetta and SWISS modeling of exemplary variants of wildtype LbCas12a are shown below.

Mutation 1, G532A: The structure of an RNP comprising the G532A variant nucleic acid-guided nuclease is shown in FIG. 11A. Modeling indicated the following changes to the wildtype LbCas12a structure with the G532A substitution (seen in FIG. 11A as a red residue): loss of one hydrogen bond with TS-PAM (target strand PAM) at amino acid residue 595; loss of one hydrogen bond with NTS-PAM (non-target strand PAM) at amino acid residue 595; no addition or loss of a hydrogen bond at amino acid residue 532. Per simulations, mutation G532A is a structurally stabilizing mutation. The parameters collected from SWISS-MODEL and Rosetta analysis are shown in Table 17.

TABLE 17 Mutation 1: G532A Global RMSD: 0.976 PI RMSD: 0.361 REC1 RMSD: 0.289 (235 to 235 atoms) WED RMSD: 0.306 (198 to 198 atoms) ΔΔG Flex Value: -1.13 PI = PAM-interacting domain of the G532A variant REC1 = REC1 domain of the G532A variant WED = WED domain of the G532A variant

Mutation 2, K538A: The structure of an RNP comprising the K538A variant nucleic acid-guided nuclease is shown at left in FIG. 11B. Modeling indicated the following changes to the wildtype LbCas12a structure with the K538A substitution (seen in FIG. 11B as a pink residue): loss of one hydrogen bond with TS-PAM (target strand PAM) at amino acid residue 538; loss of one hydrogen bond with TS-PAM (target strand PAM) at amino acid residue 595; loss of one hydrogen bond with NTS-PAM (non-target strand PAM) at amino acid residue 595. Per simulations, mutation K538A is a structurally stabilizing mutation. The parameters collected from SWISS-MODEL and Rosetta analysis are shown in Table 18.

TABLE 18 Mutation 2: K538A Global RMSD: 0.990 PI RMSD: 0.376 REC1 RMSD: 0.305 (236 to 236 atoms) WED RMSD: 0.324 (194 to 194 atoms) ΔΔG Flex Value: 0.06 PI = PAM-interacting domain of the K538A variant REC1 = REC1 domain of the K538A variant WED = WED domain of the K538A variant

Mutation 3, Y542A: The structure of an RNP comprising the Y542A variant nucleic acid-guided nuclease is shown in FIG. 11C. Modeling indicated the following changes to the wildtype LbCas12a structure with the Y542A substitution (seen in FIG. 11C as a blue residue): loss of two hydrogen bonds with TS-PAM (target strand PAM) at amino acid residue 542; loss of one hydrogen bond with TS-PAM (target strand PAM) at amino acid residue 538; loss of one hydrogen bond with TS-PAM (target strand PAM) at amino acid residue 595; loss of one hydrogen bond with NTS-PAM (non-target strand PAM) at amino acid residue 595. Per simulations, mutation Y542A is a structurally stabilizing mutation. The parameters collected from SWISS-MODEL and Rosetta analysis are shown in Table 19.

TABLE 19 Mutation 3: Y542A Global RMSD: 0.989 PI RMSD: 0.377 REC1 RMSD: 0.306 (237 to 237 atoms) WED RMSD: 0.338 (199 to 199 atoms) ΔΔG Flex Value: - 2.06 PI = PAM-interacting domain of the Y542A variant REC1 = REC1 domain of the Y542A variant WED = WED domain of the Y542A variant

Mutation 4, K595A: The structure of an RNP comprising the K595A variant nucleic acid-guided nuclease is shown in FIG. 11D. Modeling indicated the following changes to the wildtype LbCas12a structure with the K595A substitution (seen in FIG. 11D as an orange residue): loss of two hydrogen bonds with TS-PAM (target strand PAM) at amino acid residue 595; loss of one hydrogen bond with NTS-PAM (non-target strand PAM) at amino acid residue 595; loss of one hydrogen bond with NTS-PAM (non-target strand PAM) at amino acid residue 538. Per simulations, mutation K595A is a structurally destabilizing mutation. The parameters collected from SWISS-MODEL and Rosetta analysis are shown in Table 20.

TABLE 20 Mutation 4: K595A Global RMSD: 0.976 PI RMSD: 0.361 REC1 RMSD: 0.289 (235 to 235 atoms) WED RMSD: 0.306 (198 to 198 atoms) ΔΔG Flex Value: 1.26 PI = PAM-interacting domain of the K595A variant REC1 = REC1 domain of the K595A variant WED = WED domain of the K595A variant

Mutation 5, Combination G532A, K538A, Y542A, and K595A: The structure of an RNP comprising the combination G532A/K538A/Y542A/K595A variant (“combination variant”) nucleic acid-guided nuclease is shown in FIG. 11E. Modeling indicated the following changes to the wildtype LbCas12a structure with the four substitutions: loss of five hydrogen bonds with TS-PAM (target strand PAM); loss of one hydrogen bond with NTS-PAM (non-target strand PAM). Per simulations, the combination variant is structurally stable. The parameters collected from SWISS-MODEL and Rosetta analysis are shown in Table 21.

TABLE 21 Mutation 5: G532A/K538A/Y542A/K595A Global RMSD: 0.966 PI RMSD: 0.351 REC1 RMSD: 0.261 (226 to 226 atoms) WED RMSD: 0.288 (200 to 200 atoms) ΔΔG Flex Value: -3.31 PI = PAM-interacting domain of the combination variant REC1 = REC1 domain of the combination variant WED = WED domain of the combination variant

Mutation 6, K595D: The structure of an RNP comprising the K595D variant nucleic acid-guided nuclease is shown in FIG. 11F. Modeling indicated the following changes to the wildtype LbCas12a structure at location 595 with this substitution: loss of two hydrogen bonds with TS-PAM (target strand PAM); loss of one hydrogen bond with NTS-PAM (non-target strand PAM); and gain of one hydrogen bond with NTS-PAM. Per simulations, the K595D variant is structurally unstable. The parameters collected from SWISS-MODEL and Rosetta analysis are shown in Table 22.

TABLE 22 Mutation 6: K595D Global RMSD: 1.001 PI RMSD: 0.367 (89 to 89 atoms) REC1 RMSD: 0.296 (235 to 235 atoms) WED RMSD: 0.320 (197 to 197 atoms) ΔΔG Flex Value: 2.04 PI = PAM-interacting domain of the combination variant REC1 = REC1 domain of the combination variant WED = WED domain of the combination variant

Mutation 7, K595E: The structure of an RNP comprising the K595E variant nucleic acid-guided nuclease is shown in FIG. 11G. Modeling indicated the following changes to the wildtype LbCas12a structure at location 595 with this substitution: loss of two hydrogen bonds with TS-PAM (target strand PAM); loss of one hydrogen bond with NTS; and no gain of hydrogen bonds. Per simulations, the K595E variant is structurally unstable. The parameters collected from SWISS-MODEL and Rosetta analysis are shown in Table 23.

TABLE 23 Mutation 6: K595E Global RMSD: 0.975 PI RMSD: 0.352 (89 to 89 atoms) REC1 RMSD: 0.264 (226 to 226 atoms) WED RMSD: 0.290 (198 to 198 atoms) ΔΔG Flex Value: 1.37 PI = PAM-interacting domain of the combination variant REC1 = REC1 domain of the combination variant WED = WED domain of the combination variant

Mutation 8, Combination K538A, Y542A, K595D: The structure of an RNP comprising the combination K538A/Y542A/K595D variant (“combination variant”) nucleic acid-guided nuclease is shown in FIG. 11H. Modeling indicated the following changes to the wildtype LbCas12a structure with the three substitutions: loss of two hydrogen bonds with TS (target strand) at position 595; loss of one hydrogen bond with NTS (non-target); combined loss of three hydrogen bonds at 532/242 positions; and gain of one hydrogen bond at 595. Per simulations, the combination variant is structurally destabilizing. The parameters collected from SWISS-MODEL and Rosetta analysis are shown in Table 24.

TABLE 24 Mutation 6: K538A, Y542A, K595D Global RMSD: 0.976 PI RMSD: 0.351 (89 to 89 atoms) REC1 RMSD: 0.261 (225 to 225 atoms) WED RMSD: 0.289 (198 to 198 atoms) ΔΔG Flex Value: 0.96 PI = PAM-interacting domain of the combination variant REC1 = REC1 domain of the combination variant WED = WED domain of the combination variant

Mutation 9, Combination K538A, Y542A, K595E: The structure of an RNP comprising the combination K538A/Y542A/K595E variant (“combination variant”) nucleic acid-guided nuclease is shown in FIG. 11I. Modeling indicated the following changes to the wildtype LbCas12a structure with the three substitutions: loss of two hydrogen bonds with TS (target strand) at position 595; loss of one hydrogen bond with NTS (non-target); combined loss of three hydrogen bonds at 532/242 positions. Per simulations, the combination variant is structurally stabilizing. The parameters collected from SWISS-MODEL and Rosetta analysis are shown in Table 25.

TABLE 25 Mutation 6: K538A, Y542A, K595E Global RMSD: 0.976 PI RMSD: 0.351 (89 to 89 atoms) REC1 RMSD: 0.261 (225 to 225 atoms) WED RMSD: 0.289 (198 to 198 atoms) ΔΔG Flex Value: -3.71 PI = PAM-interacting domain of the combination variant REC1 = REC1 domain of the combination variant WED = WED domain of the combination variant

In addition to amino acid substitutions, modifications, such as chemical modifications, can be made to amino acids identified by the structural and homology modeling described above. FIG. 6G illustrates an exemplary scheme for acetylating amino acid residue 595 in LbCas12a, a modification which prevents unwinding of dsDNA by blocking entry of a target nucleic acid into the RNP via steric hindrance. LbCas12a is combined with AcrVA5 and the reaction is incubated for 20 minutes at room temperature, resulting in LECas 12a that has been acetylated at amino acid residue 595 (K595K^(AC)). (For a discussion and methods for disabling of Cas12a by ArVA5, see Dong, et al., Nature Structural and Molecular Bio., 26(4):308-14 (2019).) DsDNA is not a substrate for LbCas12a with a K595K^(AC) modification; however, ssDNA is a substrate for LbCas12a with a K595K^(AC) modification; thus, LbCas12a (K595K^(AC)) has the desired properties of the variant nucleic acid-guided nucleases described above. In addition to acetylation, phosphorylation and methylation of select amino acid residues may be employed.

Example VIII: Single-Strand Specificity of the Variant Nucleic Acid-Guided Nucleases

In vitro transcription/translation reactions were performed for variant LbaCas12a nucleases as noted in Table 26 using the nucleic acid sequences listed in Table 27:

TABLE 26 Template DNA for IVTT 250 ng gRNA concentration 100 nM DNA activator concentration 25 nM Probe concentration 500 nM Reaction volume 30 µL Reporter 5′-FAM-TTATTATT-IABkFQ-3′ Plate PCR plate 96-well, black Read temperature 25° C. Read duration 30 minutes Buffer NEB r2.1 New England Biolabs®, Inc., Ipswich, MA) Na+ 50 mM Mg+2 10 mM

TABLE 27 Activator RunX fragment (dsDNA + PAM) GCCTTCAGAAGAGGGTGCATTTTCAGGAGGAAGCGAT GGCTTCAGACAGCATATTTGAGTCATT (SEQ ID NO. 617) RunX fragment (dsDNA - PAM) GCCTTCAGAAGAGGGTGCATGCACAGGAGGAAGCGAT GGCTTCAGACAGCATATTTGAGTCATT (SEQ ID NO. 618) Target region in activator AGGAGGAAGCGATGGCTTCAGA (SEQ ID NO. 619) gRNA LbaCas12a gRNA gUAAUUUCUACUAAGUGUAGAUAGGAGGAAGCGAUG GCUUCAGA (SEQ ID NO. 620)

The results are shown in FIGS. 12A - 12G indicating the time for detection of dsDNA and ssDNA both with and without PAM sequences for purified wildtype LbaCas12a and three variants (K538A + K595A, K595A, and K538A + Y542 + K595A, and unpurified engineered variants of LbaCas12a: K538D + Y542A + K595D, K595D, K538A + K595D, K538A + K595E, G532A + K538A + Y542A + K595A, K538A + Y542A + K595D, K538D + Y542A + K595A, K538D + Y542D + K595A, and K538E + Y542A + K595A. Note that all variant engineered nucleic acid-guided nucleases slowed down double-strand DNA detection to varying degrees, with the double and triple variants at positions K538, Y542 and K595 of wt LbaCas12a performing best in comparison to wt LbCas12a, while single-strand DNA detection remained high, both in single-strand DNA with a PAM and without a PAM. The following variants were particularly robust: K538D + Y542A + K595D, K538A + K595D, K538A + K595E, G532A + K538A + Y542A + K595A, K538D + Y542A + K595A, and K538D + Y542D + K595D.

FIGS. 13A and 13B show the sequence alignment of many different Cas12a nucleases and orthologs, including in some instances several alignments of the same Cas12a nuclease.

Example IX: Detection of Biomarker Alpha-Synuclein in CSF for Monitoring Progression of Parkinson’s Disease

The biomarker α-synuclein, which is found in both aggregated and fibrillar form, has attracted attention as a biomarker of Parkinson’s disease. Human α-synuclein is expressed in the brain in the neocortex, hippocampus, substantia nigra, thalamus and cerebellum. It is encoded by the SNCA gene that consists of six exons ranging in size from 42 to 1110 base pairs. The predominant form of α-synuclein is the full-length protein, but other shorter isoforms exist. C-terminal truncation of α-synuclein induces aggregation, suggesting that C-terminal modifications may be involved in Parkinson’s pathology. Changes in the levels of α-synuclein have been reported in CSF of Parkinson’ patients. The gradual spread of α-synuclein pathology leads to a high concentration of extracellular α-synuclein that can potentially damage healthy neurons. Here, the cascade assay is used to monitor the level of nucleic acids in cerebrospinal fluid (CSF) to monitor the levels of mRNA transcripts that when translated lead to a truncated α-synuclein protein.

A lumbar puncture is performed on an individual, withdrawing approximately 5 mL of cerebrospinal fluid (CSF) for testing. The CSF sample is then treated by phenol-chloroform extraction or oligo dT affinity resins via a commercial kit (see, e.g., the TurboCapture mRNA kit or RNeaxy Pure mRNA Bead Kit from Qiagen®). Briefly, two RNPls are preassembled as described above in Example II with a first gRNA sequence designed to target the coding sequence of the mRNA transcribed from SNCA gene specific to the C-terminus region of α-synuclein to detect full-length α-synuclein and second gRNA sequence designed to target the coding sequence of the mRNA transcribed from SNCA gene specific to the N-terminus region of α-synuclein to detect all α-synuclein mRNAs. In addition to the gRNA, each RNP1 also comprises an LbCas13a nuclease (i.e., an RNA-specific nuclease). Also as described in Example II above, an RNP2 is preassembled with a gRNA sequence designed to target an unblocked nucleic acid molecule that results from unblocking (i.e., linearizing) a chosen blocked nucleic acid molecule such as U29. The blocked nucleic acid molecule is formed as described above in Example III, and a reporter is formed as described above in Example IV. The reaction mix contains the preassembled RNP1, preassembled RNP2, and a blocked nucleic acid molecule, in a buffer (pH of about 8) containing 4 mM MgCl₂ and 101 mM NaCl. The cascade assay is performed by one of the protocols described above in Example V. A readout is performed by comparing the level of N-terminus coding sequences detected (the level of total α-synuclein mRNA) versus the level of C-terminus coding sequences detected (the level of full-length α-synuclein mRNA).

Example X: Detection of Foot and Mouth Disease Virus From Nasal Swabs

Foot-and-mouth disease (FMD) is a severe and highly contagious viral disease. The FMD virus causes illness in cows, pigs, sheep, goats, deer, and other animals with divided hooves and is a worldwide concern as it can spread quickly and cause significant economic losses. FMD has serious impacts on the livestock trade — a single detection of FMD will stop international trade completely for a period of time. Since the disease can spread widely and rapidly and has grave economic consequences, FMD is one of the animal diseases livestock owners dread most. FMD is caused by a virus, which survives in living tissue and in the breath, saliva, urine, and other excretions of infected animals. FMD can also survive in contaminated materials and the environment for several months under the right conditions.

A nasal swab is performed on a subject, such as a cow or pig, and the nucleic acids extracted using, e.g., the Monarch Total RNA Miniprep Kit (New England Biolabs®, Inc., Ipswich, MA). Briefly, an RNP1 is preassembled as described above in Example II with a gRNA sequence designed to a gene from the FMD virus (e.g., to a portion of NCBI Reference Sequence NC_039210.1) and an LbCas12a nuclease (i.e., a DNA-specific nuclease). Also as described in Example II above, an RNP2 is preassembled with a gRNA sequence designed to target an unblocked nucleic acid molecule that results from unblocking (i.e., linearizing) a chosen blocked nucleic acid molecule such as U29. The blocked nucleic acid molecule is formed as described above in Example III, and a reporter is formed as described above in Example IV. The reaction mix contains the preassembled RNP1, preassembled RNP2, and a blocked nucleic acid molecule, in a buffer (pH of about 8) containing 4 mM MgCl₂ and 101 mM NaCl. The cascade assay is performed by one of the protocols described above in Example V, and the readout is positive detection of FMD virus-specific DNA sequences.

Example XI: Detection of Sickle Cell Gene Sequences in Peripheral Blood

Sickle cell disease (SCD) is a group of inherited red blood cell disorders. In someone who has SCD, the hemoglobin is abnormal, which causes the red blood cells to become hard and sticky and look like a C-shaped farm tool called a “sickle.” The sickle cells die early, which causes a constant shortage of red blood cells; in addition, when the sickle-shaped blood cells travel through small blood vessels, they get stuck and clog the blood flow, causing pain and other serious complications such as infection and stroke.

One form of SCD is HbSS. Individuals who have this form of SCD inherit two genes, one from each parent, that code for hemoglobin “S.” Hemoglobin S is an abnormal form of hemoglobin that causes the red cells to become rigid and sickle shaped. This is commonly called sickle cell anemia and is usually the most severe form of the disease. Another form of SCD is HbSC. Individuals who have this form of SCD inherit a hemoglobin “S” gene from one parent and a gene for a different type of abnormal hemoglobin called “C” from the other parent. This is usually a milder form of SCD. A third form of SCD is HbS thalassemia. Individuals who have this form of SCD inherit a hemoglobin “S” gene from one parent and a gene for beta thalassemia, another type of hemoglobin abnormality, from the other parent. There are two types of beta thalassemia: “zero” (HbS beta0) and “plus” (HbS beta+). Those with HbS beta0-thalassemia usually have a severe form of SCD. People with HbS beta+-thalassemia tend to have a milder form of SCD.

A non-invasive prenatal test (NIPT) that uses only maternal cell-free DNA (cfDNA) from peripheral blood permits prenatal detection of sickle cell disease and beta thalassemia by screening without the need for paternal DNA. Such a screening enables patients and healthcare providers to make informed decisions about diagnostic testing and may expand gene therapy treatment options. A 10 mL peripheral blood draw is performed on a pregnant subject into a Streck tube. The blood is treated with lysis-binding buffer and proteinase K under denaturing conditions at 55° C. for 15 minutes in the presence of magnetic beads. Following the heating step, the mixture is incubated for 1 hour at room temperature with mixing every 10 minutes at 1200 rpm for 30 seconds on an Eppendorf themomixer. The beads are captured on a magnetic stand for 2 minutes, washed three times after which cfDNA is eluted by adding elution buffer and incubating for 5 minutes at 55° C. The cfDNA is further purified by diluting in 1:1 FTA (Fast Technology for Analysis) reagent, cat# WHAWB 120204 (Sigma-Aldrich, USA), containing NaCl (sodium chloride); Tris; EDTA (ethylenediaminetetraacetic acid); TRITON-X-100 (t-Octylphenoxypolyethoxyethanol) and incubated for 10 minutes at room temperature. An additional bead purification step is performed using PCRClean DX beads, cat# C-1003-450 (ALINE Biosciences, USA). Alternatively, there are several kits available commercially that are designed to extract cfDNA including the BioChain® cfPure® Cell free DNA Extraction Kit (BioChain®, Newark, CA); the Monarch Genomic DNA Purification Kit and the Monarch HMW DNA Extraction Kit for Blood (New England Biolabs®, Inc., Ipswich, MA); and the cfDNA Purification Kit (Active Motif®, Carlsbad, CA).

For the cascade assay, three RNPls are preassembled as described above in Example II with 1) gRNA sequence designed to detect the Hemoglobin S gene variant and an LbCas12a nuclease (i.e., an DNA-specific nuclease); 2) a gRNA sequence designed to detect the Hemoglobin C gene variant and an LbCas 12a nuclease (i.e., an DNA-specific nuclease); and 3) a gRNA sequence designed to detect the gene for beta thalassemia and an LbCas12a nuclease (i.e., an DNA-specific nuclease). Also as described in Example II above, an RNP2 is preassembled with a gRNA sequence designed to target an unblocked nucleic acid molecule that results from unblocking (i.e., linearizing) a chosen blocked nucleic acid molecule such as U29. The blocked nucleic acid molecule is formed as described above in Example III, and a reporter is formed as described above in Example IV. The reaction mix contains the preassembled RNP1, preassembled RNP2, and a blocked nucleic acid molecule, in a buffer (pH of about 8) containing 4 mM MgCl₂ and 101 mM NaCl. The cascade assay is performed by one of the protocols described above in Example V. The readout is detection of the Hemoglobin S gene variant, the detection of the Hemoglobin S variant and the Hemoglobin C variant, and the detection of the Hemoglobin S variant and the β-thalassemia gene.

Example XII: Detection of Donor-Derived Gene Sequences in Peripheral Blood of Transplant Patients

Costly and invasive tissue biopsies to detect allograft rejection after transplantation have numerous limitations; however, assays based on cell-free DNA (cfDNA) — circulating fragments of DNA released from cells, tissues, and organs as they undergo natural cell death — can improve the ability to detect rejection and implement earlier changes in management of the transplanted organ. Rejection, referring to injury of a donated organ caused by the recipient’s immune system, often causes allograft dysfunction and even patient death. T-cell mediated acute cellular rejection occurs most often within the first 6 months post-transplant. Acute cellular rejection involves accumulation of CD4+ and CD8+ T-cells in the interstitial space of the allograft as the recipient’s immune system recognizes antigens on the donated organ as foreign, initiating an immune cascade that ultimately leads to apoptosis of the targeted cells. As these cells die, genomic DNA is cleaved and fragments of donor derived-cfDNA are released to join the pool of recipient cfDNA in the blood. Using cfDNA as a biomarker for acute cellular rejection is advantageous since it is derived from the injured cells of the donated organ and therefore should represent a direct measure of cell death occurring in the allograft. Further, cfDNA maintains all of the genetic features of the original genomic DNA, allowing the genetic material released from the donated organ to be differentiated from the cfDNA derived from cells of the recipient that are undergoing natural apoptosis.

For organ transplants in which the donor is male and the recipient is female, this “sex mismatch” is leveraged to calculate donor derived-cfDNA levels from within the recipient’s total cfDNA pool. Although this approach allows for confident diagnosis of rejection in the allograft, sex-mismatch between the donor and recipient is relatively infrequent and not universally applicable; thus, the presence of other genetic differences between the donor and recipient at a particular locus are leveraged to identify the origin of the circulating cfDNA. Ideally, the recipient would be homozygous for a single base (for example, AA) and at the same locus the donor would be homozygous for a different base (for example, GG). Given the genetic heterogeneity between individuals, hundreds to tens of thousands of potentially informative loci across the genome can be interrogated to distinguish donor derived-cfDNA from recipient cfDNA.

A 10 mL peripheral blood draw is performed on a transplantation subject into a Streck tube. The blood is treated with lysis-binding buffer and proteinase K under denaturing conditions at 55° C. for 15 minutes in the presence of magnetic beads. Following the heating step, the mixture is incubated for 1 hour at room temperature with mixing every 10 minutes at 1200 rpm for 30 seconds on an Eppendorf themomixer. The beads are captured on a magnetic stand for 2 minutes, washed three times after which cfDNA is eluted by adding elution buffer and incubating for 5 minutes at 55° C. The cfDNA is further purified by diluting in 1:1 FTA (Fast Technology for Analysis) reagent, cat# WHAWB 120204 (Sigma-Aldrich, USA), containing NaCl (sodium chloride); Tris; EDTA (ethylenediaminetetraacetic acid); TRITON-X-100 (t-Octylphenoxypolyethoxyethanol) and incubated for 10 minutes at room temperature. An additional bead purification step is performed using PCRClean DX beads, cat# C-1003-450 (ALINE Biosciences, USA). Also, as stated above, there are several kits available commercially that are designed to extract cfDNA including the BioChain® cfPure® Cell free DNA Extraction Kit (BioChain®, Newark, CA); the Monarch Genomic DNA Purification Kit and the Monarch HMW DNA Extraction Kit for Blood (New England Biolabs®, Inc., Ipswich, MA); and the cfDNA Purification Kit (Active Motif®, Carlsbad, CA).

For the cascade assay, several to many different RNPls are preassembled as described above in Example II with gRNA sequences designed to 1) query Y and/or X chromosome loci in sex mismatch transplantation cases; or 2) gRNA sequences designed to query various loci that are different in the genomic DNA of the recipient and the donor; along with an LbCas12a nuclease (i.e., an DNA-specific nuclease). Also as described in Example II above, an RNP2 is preassembled with a gRNA sequence designed to target an unblocked nucleic acid molecule that results from unblocking (i.e., linearizing) a chosen blocked nucleic acid molecule such as U29. The blocked nucleic acid molecule is formed as described above in Example III, and a reporter is formed as described above in Example IV. The reaction mix contains the preassembled RNP1, preassembled RNP2, and a blocked nucleic acid molecule, in a buffer (pH of about 8) containing 4 mM MgCl₂ and 101 mM NaCl. The cascade assay is performed by one of the protocols described above in Example V. The readout detects the level of donor-specific nucleic acid sequences.

Example XIII: Detection of Microbe Contamination in a Laboratory

DNA that is found in the environment is called “environmental DNA” or eDNA (e-DNA) for short, and it is formally defined as “genetic material obtained directly from environmental samples without any obvious signs of biological source material.” eDNA has been harnessed to detect rare or invasive species and pathogens in a broad range of environments. Samples are typically collected in the form of water, soil, sediment, or surface swabs. The DNA must then be extracted and purified to remove chemicals that may inhibit the cascade reaction. Surface wipe samples are commonly collected to assess microbe contamination in, e.g., a laboratory. The wipe test protocol consists of four distinct stages: removal of DNA from surfaces using absorbent wipes, extraction of DNA from the wipes into a buffer solution, purification of DNA, and analysis of the extract.

For sample collection, sterile 2x2 inch polyester-rayon non-woven wipes are used to wipe down an environmental surface, such as a laboratory bench. Each wipe is placed into a sterile 50 ml conical tube and 10 mL of PBST is transferred to each conical tube using a sterile serological pipette. The tubes are vortexed at the maximum speed for 20 minutes using a Vortex Genie 2. A 200 µL aliquot of the supernatant was processed using a nucleic acid purification kit (QIAmp DNA Blood Mini Kit, QIAGEN, Inc., Valencia, CA). The kit lyses the sample, stabilizes and binds DNA to a selective membrane, and elutes the DNA sample.

For the cascade assay, several to many different RNPls are preassembled as described above in Example II with gRNA sequences designed to detect, e.g., Aspergillus acidus; Parafilaria bovicola; Babesia divergens; Escherichia coli; Pseudomonas aeruginosa; and Dengue virus; along with an LbCas12a nuclease (i.e., an DNA-specific nuclease). Also as described in Example II above, an RNP2 is preassembled with a gRNA sequence designed to target an unblocked nucleic acid molecule that results from unblocking (i.e., linearizing) a chosen blocked nucleic acid molecule such as U29. The blocked nucleic acid molecule is formed as described above in Example III, and a reporter is formed as described above in Example IV. The reaction mix contains the preassembled RNP1, preassembled RNP2, and a blocked nucleic acid molecule, in a buffer (pH of about 8) containing 4 mM MgCl₂ and 101 mM NaCl. The cascade assay is performed by one of the protocols described above in Example V. The readout is detection of a genomic sequence unique to a pathogen.

While certain embodiments have been described, these embodiments have been presented by way of example only and are not intended to limit the scope of the present disclosures. Indeed, the novel methods, apparatuses, modules, instruments and systems described herein can be embodied in a variety of other forms; furthermore, various omissions, substitutions and changes in the form of the methods, apparatuses, modules, instruments and systems described herein can be made without departing from the spirit of the present disclosures. The accompanying claims and their equivalents are intended to cover such forms or modifications as would fall within the scope and spirit of the present disclosures. 

We claim:
 1. A method for preventing unwinding of blocked nucleic acid molecules in the presence of an RNP comprising the steps of: providing blocked nucleic acid molecules; providing ribonucleoprotein complexes comprising a nucleic acid-guided nuclease that exhibits both cis- and trans-cleavage activity upon activation and a gRNA that recognizes an unblocked nucleic acid molecule resulting from trans-cleavage of the blocked nucleic acid molecules; and one of 1) providing blocked nucleic acid molecules and ribonucleoprotein complexes where the molar ratio of the blocked nucleic acid molecules equals or exceeds the molar ratio of the ribonucleoprotein complexes; 2) engineering the nucleic acid-guided nuclease used in the ribonucleoprotein complex to result in a variant nucleic acid-guided nuclease such that single stranded DNA is cleaved faster than double stranded DNA is cleaved; and/or 3) engineering the blocked nucleic acid molecules to include bulky modifications of a size of approximately 1 nm or less.
 2. The method of claim 1, wherein blocked nucleic acid molecules include bulky modifications and the bulky modification is approximately 0.7 nm in size.
 3. The method of claim 2, wherein blocked nucleic acid molecules include bulky modifications and wherein the bulky modification is approximately 0.5 nm in size.
 4. The method of claim 3, wherein blocked nucleic acid molecules include bulky modifications and wherein the bulky modification is approximately 0.2 nm in size.
 5. The method of claim 1, wherein blocked nucleic acid molecules include bulky modifications and wherein there are two bulky modifications with one bulky modification located on the 5′ end of the blocked nucleic acid molecule and one bulky modification located on the 3′ end of the blocked nucleic acid molecule, and where the 5′ and 3′ ends comprising the two bulky modifications are less than 11 nm from one another.
 6. The method of claim 1, wherein blocked nucleic acid molecules include bulky modifications and wherein the bulky modification is on a 5′ end of blocked nucleic acid molecules.
 7. The method of claim 6, wherein the bulky modification is selected from the group of 5′ Fam (6-fluorescein amidite); Black Hole Quencher-1-5′; biotin TEG (15 atom triethylene glycol spacer); biotin-5′; and cholesterol TEG (15 atom triethylene glycol spacer).
 8. The method of claim 1, wherein blocked nucleic acid molecules include bulky modifications and wherein the bulky modification is on a 3′ end of the blocked nucleic acid molecules.
 9. The method of claim 8, wherein the bulky modification is selected from the group of Black Hole Quencher-1-3′; biotin-3′; and TAMRA-3′ (carboxytetramethylrhodamine).
 10. The method of claim 1, wherein blocked nucleic acid molecules include bulky modifications and wherein the bulky modification is between two internal nucleic acid residues of the blocked nucleic acid molecules.
 11. The method of claim 10, wherein the bulky modification is selected from the group of Cy3 internal and Cy5 internal.
 12. The method of claim 1, wherein blocked nucleic acid molecules include bulky modifications and wherein the bulky modification is an internal nucleotide base modification.
 13. The method of claim 12, wherein the bulky modification is selected from the group of biotin deoxythymidine dT; disthiobiotin NHS; and fluorescein dT.
 14. The method of claim 1, wherein the blocked nucleic acid molecules comprise a structure represented by any one of Formulas I-IV, wherein Formulas I-IV are in the 5′-to-3′ direction: (a) A-(B-L)_(J)-C-M-T-D (Formula I); wherein A is 0-15 nucleotides in length; B is 4-12 nucleotides in length; L is 3-25 nucleotides in length; J is an integer between 1 and 10; C is 4-15 nucleotides in length; M is 1-25 nucleotides in length or is absent, wherein if M is absent then A-(B-L)_(J)-C and T-D are separate nucleic acid strands; T is 17-135 nucleotides in length and comprises at least 50% sequence complementarity to B and C; and D is 0-10 nucleotides in length and comprises at least 50% sequence complementarity to A; (b) D-T-T′-C-(L-B)_(J)-A (Formula II); wherein D is 0-10 nucleotides in length; T-T′ is 17-135 nucleotides in length; T′ is 1-10 nucleotides in length and does not hybridize with T; C is 4-15 nucleotides in length and comprises at least 50% sequence complementarity to T; L is 3-25 nucleotides in length and does not hybridize with T; B is 4-12 nucleotides in length and comprises at least 50% sequence complementarity to T; J is an integer between 1 and 10; A is 0-15 nucleotides in length and comprises at least 50% sequence complementarity to D; (c) T-D-M-A-(B-L)_(J)-C (Formula III); wherein T is 17-135 nucleotides in length; D is 0-10 nucleotides in length; M is 1-25 nucleotides in length or is absent, wherein if M is absent then T-D and A-(B-L)_(J)-C are separate nucleic acid strands; A is 0-15 nucleotides in length and comprises at least 50% sequence complementarity to D; B is 4-12 nucleotides in length and comprises at least 50% sequence complementarity to T; L is 3-25 nucleotides in length; J is an integer between 1 and 10; and C is 4-15 nucleotides in length; or (d) T-D-M-A-L_(p)-C (Formula IV); wherein T is 17-31 nucleotides in length (e.g., 17-100, 17-50, or 17-25); D is 0-15 nucleotides in length; M is 1-25 nucleotides in length; A is 0-15 nucleotides in length and comprises a sequence complementary to D; and L is 3-25 nucleotides in length; p is 0 or 1; C is 4-15 nucleotides in length and comprises a sequence complementary to T.
 15. The method of claim 1, wherein the RNP comprises a variant nucleic acid-guided nuclease and wherein the variant nucleic acid-guided nuclease is a Type V variant nucleic acid-guided nuclease or a Type VI variant nucleic acid-guided nuclease.
 16. The method of claim 1, wherein the RNP comprises a variant nucleic acid-guided nuclease comprising at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecule and wherein the mutation is selected from mutations to amino acid residues K538, Y542 and K595 in relation to SEQ ID NO: 1 and equivalent amino acid residues in orthologs.
 17. The method of claim 16, wherein there are at least two mutations to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules selected from mutations to amino acid residues K538, Y542 and K595 in relation to SEQ ID NO: 1 and equivalent amino acid residues in orthologs.
 18. The method of claim 17, wherein there are at least three mutations to domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecule selected from mutations to amino acid residues K538, Y542 and K595 in relation to SEQ ID NO: 1 and equivalent amino acid residues in orthologs.
 19. The method of claim 1, wherein the RNP comprises a variant nucleic acid-guided nuclease comprising at least one mutation to domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecule and wherein the at least one mutation is selected from mutations to amino acid residues K548, N552 and K607 in relation to SEQ ID NO:2; mutations to amino acid residues K534, Y538 and R591 in relation to SEQ ID NO:3; mutations to amino acid residues K541, N545 and K601 in relation to SEQ ID NO:4; mutations to amino acid residues K579, N583 and K635 in relation to SEQ ID NO:5; mutations to amino acid residues K613, N617 and K671 in relation to SEQ ID NO:6; from mutations to amino acid residues K613, N617 and K671 in relation to SEQ ID NO:7; mutations to amino acid residues K617, N621 and K678 in relation to SEQ ID NO:8; mutations to amino acid residues K541, N545 and K601 in relation to SEQ ID NO:9; mutations to amino acid residues K569, N573 and K625 in relation to SEQ ID NO:10; mutations to amino acid residues K562, N566 and K619 in relation to SEQ ID NO:11; mutations to amino acid residues K645, N649 and K732 in relation to SEQ ID NO: 12; mutations to amino acid residues K548, N552 and K607 in relation to SEQ ID NO:13; mutations to amino acid residues K592, N596 and K653 in relation to SEQ ID NO: 14; or mutations to amino acid residues K521, N525 and K577 in relation to SEQ ID NO:
 15. 20. The method of claim 1, wherein the RNP comprises a variant nucleic acid-guided nuclease comprising at least one mutation to domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecule and wherein single stranded DNA is cleaved at least two times faster than double stranded DNA is cleaved.
 21. The method of claim 21, wherein the RNP comprises a variant nucleic acid-guided nuclease comprising at least one mutation to domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecule and wherein single stranded DNA is cleaved at least three times faster than double stranded DNA is cleaved.
 22. The method of claim 22, wherein the RNP comprises a variant nucleic acid-guided nuclease comprising at least one mutation to domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecule and wherein single stranded DNA is cleaved at least four times faster than double stranded DNA is cleaved.
 23. The method of claim 1, wherein the RNP comprises a variant nucleic acid-guided nuclease and wherein the variant nucleic acid-guided nuclease is engineered such that single stranded DNA is cleaved faster than double stranded DNA is cleaved, wherein the variant nuclease comprises at least two mutations to domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecule and wherein the variant nuclease exhibits both cis- and trans-cleavage activity.
 24. The method of claim 23, wherein the RNP comprises a variant nucleic acid-guided nuclease and wherein the variant nucleic acid-guided nuclease is engineered such that single stranded DNA is cleaved faster than double stranded DNA is cleaved, wherein the variant nuclease comprises at least three mutations to domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecule and wherein the variant nuclease exhibits both cis- and trans-cleavage activity.
 25. The method of claim 1, wherein the plurality of blocked nucleic acid molecules and the RNP2s are at a concentration ratio of at least 2 blocked nucleic acids to 1 RNP2 in the reaction mixture.
 26. The method of claim 25, wherein the plurality of blocked nucleic acid molecules and the RNP2s are at a concentration ratio of at least 3 blocked nucleic acids to 1 RNP2 in the reaction mixture.
 27. The method of claim 26, wherein the plurality of blocked nucleic acid molecules and the RNP2s are at a concentration ratio of at least 4 blocked nucleic acids to 1 RNP2 in the reaction mixture.
 28. The method of claim 27, wherein the plurality of blocked nucleic acid molecules and the RNP2s are at a concentration ratio of at least 5 blocked nucleic acids to 1 RNP2 in the reaction mixture.
 29. The method of claim 1, wherein the plurality of blocked nucleic acid molecules and the RNP2s are at a concentration ratio of at least 1.5 blocked nucleic acids to 1 RNP2 in the reaction mixture.
 30. The method of claim 1, wherein the RNP comprises a variant nucleic acid-guided nuclease and wherein the variant nucleic acid-guided nuclease has a sequence selected from the group of SEQ ID NO: 16 to SEQ ID NO:
 600. 